Compositions and methods for treating liver disease

Abstract

To provide a composition for treating a liver disease in a subject by increasing transport or retention of HNF4α, a transcription factor, into the nucleus of hepatocytes in the subject.SOLUTION: Provided is a pharmaceutical composition for treating a liver disease in a subject in need thereof, wherein the composition decreases the amount or suppresses the function of one or more transcription factors selected from the group consisting of DNAJB1 / HSP40, ATF6, ATF4, and PERK.SELECTED DRAWING: Figure 16

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application was filed on October 16, 2019, which is incorporated herein by reference in its entirety. Priority is claimed to filed U.S. Provisional Patent Application No. 62 / 915,765. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made under grant number DK099257 awarded by the National Institutes of Health. The Government has certain rights in this invention. The present disclosure relates to, for example, methods for controlling the expression or activity of HNF4α to treat liver disease and / or liver injury. or modulation of activity. [Background technology]

[0002] End-stage liver failure (TLF), a result of advanced cirrhosis, was the leading cause of death in 2015. It is reported to be 12th in the world with 15.8 deaths per 100,000 people. In the United States, the number of chronic liver disease cases in 2015 was estimated to be 1.2% (Tsochatzis EA et al., 2014). The number of registered deaths linked to liver disease and cirrhosis was 40,326, which is 100,000 per 100,000 people. The annual death toll was 12.5, with an annual increase rate of 3.8% (Murphy SL et al., 2017; Go ldman L, et al., 2016). The most affected age group is 45-64 years old, with a population of 100,000. With 26.4 deaths per year, chronic liver disease and cirrhosis are the leading cause of death in the United States, behind cancer, heart disease, and It ranks as the fourth leading cause of death in this age group, after accidents. Murphy SL et al., 2017). The only definitive treatment for TLF is orthotopic liver transplantation. Given the number of patients needing transplants and the insufficient number of organs available, TLF is an incurable disease (Lopez PM et al., 2006).

[0003] Chronic liver diseases include chronic infection with hepatitis viruses, alcohol-mediated cirrhosis, and non-alcoholic liver diseases. There are various causes, including nonalcoholic steatohepatitis (NASH) (Archambeaud I et al., 2015;Donato F et al,. 2006;Gelatti U et al., 2005;Kuper H et al., 2000), Each of these may lead to hepatocellular failure (Guzman-Lepe J et al., 2018; Hernaez R et al., 2017;Lee YA et al., 2015;Pessayre D et al., 1978). Human hepatocytes The mechanisms responsible for the decline in function and ultimately liver failure are poorly understood.

[0004] The major causes of chronic liver disease, cirrhosis, and recently TLF are hepatitis B and C viruses. infection, alcohol-mediated Laennec's cirrhosis, and nonalcoholic steatohepatitis It has been implicated in nonalcoholic steatohepatitis (NASH) / metabolic syndrome (Archambeaud et al., 2015; Donato F et al., 2006; Gelatti et al., 2005; Kuper et al., 2000). These etiological factors are , causing fibrosis that disrupts normal lobule architecture accompanied by alterations in the vasculature (Goldman L, et These pathological changes are associated with hepatocellular failure and the inability of hepatocytes to perform their normal functions. Although it has been associated with inability to fulfill one's responsibilities (Guzman-Lepe J et al., 2018; Hernaez R et al., 2007;Lee Ya et al., 2015;Pessayre D et al., 1978), leading to a decline in hepatocellular function and The mechanisms responsible for the progression of liver damage and eventual liver failure are unknown in humans. Tres (Cichoz-Lach H et al., 2014; Simoes ICM et al., 2018) and endoplasmic reticulum stress These induce cell death (Malhi H et al., 2011; Zhang XQ et al., 2014). (Cichoz-Lach H et al., 2014;Malhi H et al., 2011;Zhang XQ et al., 2014;W ang K et al., 2014;Seki E et al., 2015), ultimately reducing the proliferation capacity of hepatocytes. (Zhang BH et al., 1999; Michalopoulos GK et al., 2015; Dubuquoy L et al., 2015 ).

[0005] Liver-enriched transcription factors are stably downregulated in hepatocytes from rats with end-stage liver cirrhosis. It has been rated (Nishikawa T et al., 2014; Guzman-Lepe J et al., 2019), and Furthermore, forced re-expression of one of these, hepatocyte nuclear factor 4 alpha (HNF4α), reprogram dysfunctional hepatocytes to restore function both in culture and in vivo It has been shown to improve liver function in a large number of patients with advanced liver disease (Nishikawa T et al., 2014). In a study of a similar cohort, the level of HNF4α mRNA expression in diseased liver was It correlates with the degree of liver dysfunction (Child-Pugh classification) and HNF4α expression. It has been revealed that it was not localized in the nucleus (Guzman-Lepe J et al., 2019). Forced re-expression of hepatocyte nuclear factor 4 alpha (HNF4α) induces the production of new hepatocytes or stem cells dysfunctional hepatocytes both in culture and in vivo without expanding the It can be reprogrammed to make end-stage cirrhotic livers function again (Nishikawa T et al., 2014). LE, including a significant decrease in the nuclear localization and mRNA expression of HNF4α. Downregulation of TF is a key factor in hepatic function in a large cohort of human livers with TLF. It is related to the degree of disability (Guzman-Lepe J et al., 2018).

[0006] HNF4α is a transcription factor that plays an important role in liver organogenesis and hepatocyte function in the adult liver. It is a major HNF4α factor (Nishikawa T et al., 2014; Babeu JP et al., 2014). Actions are directed at specific target genes involved in lipid, glucose, xenobiotic, and drug metabolism. (Nishikawa T et al., 2014; Babeu JP et al., 2014). The gene encodes HNF4α (Kritis AA et al., 1999), which is involved in two different These promoters are controlled by two isoforms: It generates classes P1 and P2 (Babeu JP et al., 2014). The P1 isoform is , is expressed primarily in the adult liver, whereas the P2 isoform is expressed in the liver during embryonic development and It has been detected in the liver under pathological conditions such as cancer (Babeu JP et al., 2014; Walesky C et al., 2015; Tanaka T et al., 2006). The expression and function of HNF4α are regulated at multiple levels. controlled (Chellappa K et al., 2012;Guo H et al., 2014;Hong YH et al., 20 03;Lu H et al., 2016, Song Y et al., 2015;Soutoglou E et al., 2000;Sun K et a l., 2007;Xu Z et al., 2007;Yokoyama A et al., 2011;Zhou W et al., 2012). Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, modulating the expression of HNF4α and / or treating liver disease and / or There is a need for compositions and methods for treating liver damage. The disclosed compositions and methods address these and other needs. [Means for solving the problem]

[0008] The compositions and methods disclosed herein address certain unmet medical needs in the treatment of liver disease. In some embodiments, the present invention addresses unmet needs in patients with liver disease and / or liver cirrhosis. Or a composition for a medicine for treating liver damage and its use, wherein the composition comprises: PROX1, NR5A2, NR0B2, MTF1, SREBP1, EP300 and / or or POM121C, or POM121C, or increase DNAJB1 / HSP40, ATF6, ATF4 and PER or decreasing the amount of one or more transcription factors selected from the group consisting of K In some embodiments, compositions and uses thereof are disclosed that inhibit the function of The composition is a vector, and the vector is a vector containing PROX1, NR5A2, NR0B2, MTF1, Containing one or more of SREBP1, EP300, and / or POM121C In some embodiments, the composition comprises one or more nucleic acids encoding HNF4α. (e.g., HNF4α isoform 2). The compositions and methods disclosed herein result in a surprising increase in the amount of HNF4α in liver cells (e.g., For example, an increase in the total amount of HNF4α in hepatocytes and / or an increase in the amount of HNF4α in the nuclei of hepatocytes. This results in an increase in liver function, leading to effective treatment of liver disease (e.g., end-stage liver disease).

[0009] In some aspects, the present invention provides a method for treating liver disease in a subject in need thereof. The method includes administering a composition to a subject, the composition comprising: , NR0B2, MTF1, SREBP1, EP300, and POM121C and increasing the amount or function of one or more transcription factors selected from the group consisting of: are.

[0010] In some aspects, the present invention provides a method for treating liver disease in a subject in need thereof. Use of a composition for preparing a medicament for administering the composition to a subject. , the composition is PROX1, NR5A2, NR0B2, MTF1, SREBP1, EP30 0, and POM121C, or The use of the compound has been disclosed to increase function.

[0011] In some embodiments, the composition is a vector, and the vector is a vector encoding PROX1, NR5 Among A2, NR0B2, MTF1, SREBP1, EP300, and POM121C In some embodiments, the nucleic acid sequence comprises one or more nucleic acids encoding one or more of: The vector may contain one or more nucleic acids encoding PROX1 and / or SREBP1. The one or more nucleic acids may be DNA or mRNA.

[0012] In some aspects, the present invention provides a method for treating liver disease in a subject in need thereof. The method includes administering a composition to a subject, the composition comprising: 0, ATF6, ATF4, and PERK. Methods are disclosed for reducing the amount of or inhibiting the function of a transcription factor.

[0013] In some aspects, provided herein are methods for treating liver disease in a subject in need thereof. Use of a composition for preparing a medicament for treating a patient, the use comprising administering the composition to the patient. , wherein the composition consists of DNAJB1 / HSP40, ATF6, ATF4, and PERK. The use of a transcription factor-binding protein (TGF-binding protein) is disclosed. It is being done.

[0014] In some embodiments, administering the composition increases the amount of HNF4α in the nuclei of hepatocytes in the subject. In some embodiments, administration of the composition increases the total amount of HNF4α in hepatocytes. In some embodiments, administration of the composition does not increase the total amount of HNF4α in hepatocytes. Increase.

[0015] In some embodiments, the vector further comprises a nucleic acid encoding HNF4α. In some embodiments, the method includes administering to the subject a vector comprising a nucleic acid encoding HNF4α. In one example, the nucleic acid further comprises administering do.

[0016] In some aspects, provided herein are compositions comprising a vector, wherein the vector is P ROX1, NR5A2, NR0B2, MTF1, SREBP1, EP300, and PO M121C, and one or more transcription factors selected from the group consisting of functional fragments thereof. Disclosed are compositions comprising one or more nucleic acids encoding the offspring.

[0017] In some aspects, the present invention provides a method for treating liver disease in a subject in need thereof. The method comprises administering to a subject a vector comprising a nucleic acid encoding HNF4α isoform 2. A method is disclosed that includes providing

[0018] In some aspects, the present invention provides a method for preparing a medicament for treating liver disease. Use of a vector, wherein the vector contains a nucleic acid encoding HNF4α isoform 2. Yes, the use is disclosed. [Brief explanation of the drawings]

[0019] [Figure 1]Figures 1A-1C show the localization of HNF4α in hepatocytes from normal and cirrhotic livers. Figure 1A shows representative photographs of HNF4α immunofluorescence of isolated human hepatocytes from NASH-decompensated livers and normal human hepatocytes. Western blot analysis and quantification of HNF4α were normalized to beta-actin in hepatocytes isolated from functionally decompensated livers (NASH and alcohol-mediated Laennec cirrhosis) (n = 6) and hepatocytes isolated from normal liver controls (n = 2). Figure 1A shows total HNF4α; normal human hepatocytes vs. decompensated human hepatocytes, P = 0.166. Figure 1B shows cytoplasmic HNF4α; normal human hepatocytes vs. decompensated human hepatocytes, P = 0.023. Figure 1C shows nuclear HNF4α; normal human hepatocytes vs. decompensated human hepatocytes, P = 0.023. Graphs A-C are plotted as mean ± SD. Statistically significant (P<0.05). Diamonds indicate Child-Pugh "B" and squares indicate Child-Pugh "C." [Figure 2-1] Figures 2A-2C show the protein expression of HNF4α post-translational modifiers and Spearman's rank correlation test. Figures 2A and 2B show the expression of EGFR (P = 0.904), cMET (P = 0.023), total AMPKα (P > 0.999), p-AMPKα (Thr172) (P = 0.547), total AKT (P = 0.047), and p-AKT (S) in hepatocytes from decompensated NASH (n = 4), alcohol-mediated Laennec cirrhosis (n = 2), and normal control hepatocytes (n = 2). Western blot analysis (2A) and quantification (2B) of p-AKT(Ser473) (P = 0.547), p-AKT(Thr308) (P = 0.024), the ratio of p-AMPKα(Thr172) / AMPKα (P = 0.5476), the ratio of p-AKT(Ser473) / AKT (P = 0.1667), and the ratio of p-AKT(Thr308) / total AKT (P = 0.0238) are shown. [Figure 2-2]Figures 2A-2C show the protein expression of HNF4α post-translational modifiers and Spearman's rank correlation test. Figures 2A and 2B show the expression of EGFR (P = 0.904), cMET (P = 0.023), total AMPKα (P > 0.999), p-AMPKα (Thr172) (P = 0.547), total AKT (P = 0.047), and p-AKT (S) in hepatocytes from decompensated NASH (n = 4), alcohol-mediated Laennec cirrhosis (n = 2), and normal control hepatocytes (n = 2). Western blot analysis (2A) and quantification (2B) of p-AKT(Ser473) (P = 0.547), p-AKT(Thr308) (P = 0.024), the ratio of p-AMPKα(Thr172) / AMPKα (P = 0.5476), the ratio of p-AKT(Ser473) / AKT (P = 0.1667), and the ratio of p-AKT(Thr308) / total AKT (P = 0.0238) are shown. In Figure 2B, the filled square indicates Child-Pugh "B" and the filled diamond indicates Child-Pugh "C." [Figure 2-3] Figures 2A-2C show the protein expression of HNF4α post-translational modifiers and Spearman's rank correlation test. Figure 2C shows Spearman's rank correlation test, demonstrating that nuclear HNF4α was significantly correlated with cMET (r = 0.71; P = 0.037), total AKT (r = 0.71; P = 0.037), phospho-AKT (Thr308) (r = 0.82; P = 0.011), and the ratio of phospho-AKT (Thr308) / total AKT (r = 0.73; P = 0.031). Cytoplasmic HNF4α showed significant correlations with cMET (r = -0.80; P = 0.014), total AKT (r = -0.73; P = 0.031), phospho-AKT(Thr308) (r = -0.77; P = 0.021), and the ratio of phospho-AKT(Thr308) / total AKT (r = -0.72; P = 0.037). Bar graphs in Figure 2B are plotted as mean ± SD. Statistically significant (P < 0.05). [Figure 3-1]Figures 3A-3D show the relationship between post-translational modifiers and HNF4α cellular localization. Figure 3A shows that path analysis revealed significant direct relationships between HNF4α localization and cMET (0.56; P = 0.004), the ratio of phospho-AKT (Thr308) / total AKT (0.05; P = 0.006), and total HNF4α levels (0.60; P = 0.042). Path analysis also revealed a significant negative relationship between cMET and total HNF4α (-0.37; P = 0.024). Figure 3B depicts that linear regression analysis showed a significant relationship between nuclear HNF4α expression and the degree of liver dysfunction (Child-Pugh score) (R2 = 0.80, P = 0.007). [Figure 3-2] Figures 3A-3D show the relationship between post-translational modifiers and HNF4α cellular localization. Figure 3C illustrates that principal component analysis (PCA) revealed that protein profiles correlated with HNF4α expression positively described the characteristics of normal human hepatocytes (n = 2), while cytoplasmic HNF4α, active caspase 3, p-AKT (Ser473) / total AKT ratio, and p-AMPK (Ser172) / AMPK showed characteristics of decompensated human hepatocytes from livers with NASH (n = 4) and alcohol-mediated Laennec cirrhosis (n = 2). [Figure 3-3] Figures 3A-3D show the relationship between post-translational modifiers and HNF4α cellular localization. The graphs in Figure 3D show the fold changes in protein expression used in PCA analysis for total HNF4α, nuclear HNF4α, or cytoplasmic HNF4α (top three graphs), cMET, p-AMPK (Ser172) / AMPK, p-AKT (Ser473) / total AKT, p-AKT (Ser473) / total AKT, and phospho-AKT (Thr308) / total AKT ratios, p-H3 (Ser10), and active caspases (second and bottom graphs) in decompensated human hepatocytes (Child-Pugh classifications B and C) compared with normal human hepatocytes. Graphs are plotted as mean ± SD. Statistically significant (P<0.05). [Figure 4]Figures 4A-4C show that nuclear HNF4α acetylation is altered in human decompensated hepatocytes from explanted livers with NASH and alcohol-mediated Laennec cirrhosis. Figures 4A and 4B show Western blot and quantification of the acetylated form of HNF4α (Lys106) in the nuclear fraction of human hepatocytes from explanted livers with decompensated NASH (n = 4) and alcohol-mediated Laennec cirrhosis (n = 4) (P = 0.024). Linear regression analysis in Figure 4C shows a significant correlation between reduced acetylated form of HNF4α (Lys106) and liver dysfunction (R = 0.71, P = 0.004). Bar graphs are plotted as mean ± SD. Statistically significant (P < 0.05). [Figure 5] Figure 5 shows an in silico analysis of HNF4α-post-translational modifications (PTMs). Figure 5 provides a list of HNF4α-PTMs. [Figure 6] Figures 6A-6B show the relationship between activated AKT pathway and HNF4α post-translational modifier and p-EGFR expression in human decompensated hepatocytes from explanted livers of NASH and alcohol-mediated Laennec cirrhosis. Figure 6A shows Spearman's correlation for phospho-AKT (Thr308) / AKT. Figure 6B shows Spearman's correlation for phospho-AKT (Ser473) / AKT. [Figure 7-1] Figures 7A-7C show the expression levels of HNF4α, a liver-enriched transcription factor, in human hepatocytes isolated from explanted livers of patients with alcoholic hepatitis at the mRNA level (Figure 7A) and protein level (Figure 7B) compared with those of freshly isolated normal human hepatocytes by immunohistochemistry, demonstrating that only approximately 40% of alcoholic hepatitis hepatocytes expressed HNF4α at weak intensity in the nucleus, and approximately 10% of those cells had cytoplasmic expression of HNF4α. [Figure 7-2]Referring to Figure 7C, freshly isolated human alcoholic hepatitis hepatocytes were treated with lentivirus encoding HNF4α (Systems Bioscience, catalog number CS970S-1; HNF4α in CD511B-1). This figure shows that after 72 hours, HNF4α expression did not change the percentage of hepatocytes expressing HNF4α in the nucleus. However, HNF4α expression intensity dramatically increased in existing cells. Overall, this data indicates that HNF4α transport to the nucleus may play an important role in hepatocyte function recovery in humans with alcoholic hepatitis. [Figure 8-1] Figures 8A-8F show MTF1 expression in primary human hepatocytes. Figure 8A shows that primary human hepatocytes isolated from livers of patients undergoing liver transplantation for NASH or alcohol-induced cirrhosis were analyzed by Western blot for the expression of MTF1 (MA5-26738 1:1000) and HNF4α (ab41898 1:1000). [Figure 8-2] Figures 8A-8F show MTF1 expression in primary human hepatocytes. Figures 8B and 8C show the relative intensities of HNF4α (Figures 8B and 8C) and MTF1 (Figures 8D and 8E) between control, child B, and child C hepatocytes, compared by Brown-Forsythe one-way ANOVA and Welch's ANOVA test for multiple comparisons. HNF4α and MTF1 expression were lower in child C hepatocytes compared to child B hepatocytes. Figures 8B and 8D, *p<0.003, **p<0.01, ** *p<0.0001, n=25. Figure 8C R2=0.019, p=0.06, n=19. Figure 8E R2=0.015, p=0.1, n=19. [Figure 8-3] Figures 8A-8F show MTF1 expression in primary human hepatocytes. Figure 8F shows a correlation study using Child-Pugh score, HNF4α, and MTF1 protein expression, performed using simple linear regression. Black circles refer to controls, light gray circles refer to child B, and dark gray circles refer to child C. HNF4α protein expression correlates with MTF1 protein expression (R2 = 0.28, p = 0.007, n = 25). [Figure 9-1] Figures 9A-9D show NR0B2 expression in primary human hepatocytes. Figure 9A shows that primary human hepatocytes isolated from livers of patients undergoing liver transplantation for NASH or alcohol-induced cirrhosis were analyzed by Western blot for the expression of NR0B2 (Abclonal A1836 1:500) and HNF4α (ab41898 1:1000). [Figure 9-2] Figures 9A-9D show NR0B2 expression in primary human hepatocytes. Figures 9B and 9C show the relative intensity of NR0B2 among control, child B, and child C hepatocytes, compared by Brown-Forsythe one-way ANOVA and Welch's ANOVA test for multiple comparisons. Figure 9B: *p<0.05, **p<0.01, ***p<0.001, n=25. Figure 9C: R2=0.19, p=0.06, n=19. NR0B2 expression differs among child C, child B, and control hepatocytes. [Figure 9-3] Figures 9A-9D show NR0B2 expression in primary human hepatocytes. Figure 9D shows a correlation study using Child-Pugh score, HNF4α, and NR0B2 protein expression, performed using simple linear regression. Black circles refer to controls, light gray circles refer to child B, and dark gray circles refer to child C (R2 = 0.12, p = 0.1, n = 25). [Figure 10-1] Figures 10A-10D show NR5A2 expression in primary human hepatocytes. Figure 10A shows that primary human hepatocytes isolated from livers of patients undergoing liver transplantation for NASH or alcohol-induced cirrhosis were analyzed by Western blot for the expression of NR5A2 (Novus NBP2-27196 1:500) and HNF4α (ab41898 1:1000). [Figure 10-2]Figures 10A-10D show NR5A2 expression in primary human hepatocytes. Figures 10B and 10C show the relative intensities of NR5A2 among control, child B, and child C hepatocytes, compared by Brown-Forsythe one-way ANOVA and Welch's ANOVA test for multiple comparisons. Figure 10B *p<0.05, n=25. Figure 10C R2=0.17, p=0.07, n=19. NR5A2 expression differs between child B and child C hepatocytes and control hepatocytes. [Figure 10-3] Figures 10A-10D show NR5A2 expression in primary human hepatocytes. Figure 10D shows a correlation study using Child-Pugh score, HNF4α, and NR5A2 protein expression, performed using simple linear regression. NR5A2 protein expression correlates with HNF4α expression. Black circles indicate controls, light gray circles indicate child B, and dark gray circles indicate child C (R2 = 0.17, p < 0.05, n = 25). [Figure 11-1] Figures 11A-11D show Prox1 expression in primary human hepatocytes. Figure 11A shows that primary human hepatocytes isolated from livers of patients undergoing liver transplantation for NASH or alcohol-induced cirrhosis were analyzed by Western blot for the expression of PROX1 (R&D AF2727 1:500) and HNF4α (ab41898 1:1000). [Figure 11-2] Figures 11A-11D show Prox1 expression in primary human hepatocytes. Figures 11B and 11C show the relative intensity of PROX1 between control, child B, and child C hepatocytes compared by Brown-Forsythe one-way ANOVA and Welch's ANOVA test for multiple comparisons. Figure 11B *p<0.02, n=25. Figure 11C R2=0.02, p=0.6, n=19. PROX1 expression differs between child C and control hepatocytes. [Figure 11-3]Figures 11A-11D show Prox1 expression in primary human hepatocytes. Figure 11D shows a correlation study using Child-Pugh score, HNF4α, and protein expression of PROX1 performed using simple linear regression. Black circles refer to controls, light gray circles refer to child B, and dark gray circles refer to child C (R2 = 0.02, p = 0.46, n = 25). [Figure 12-1] Figures 12A-12D show POM121C expression in primary human hepatocytes. Figure 12A shows that primary human hepatocytes isolated from livers of patients undergoing liver transplantation for NASH or alcohol-induced cirrhosis were analyzed by Western blot for the expression of POM121C (PA5-85161 1:500) and HNF4α (ab41898 1:1000). [Figure 12-2] Figures 12A-12D show POM121C expression in primary human hepatocytes. Figures 12B and 12C show the relative intensity of POM121C among control, child B, and child C hepatocytes, compared by Brown-Forsythe one-way ANOVA and Welch's ANOVA test for multiple comparisons. Figure 12B: n=25. Figure 12C: R2=0.08, p=0.24, n=25. [Figure 12-3] Figures 12A-12D show POM121C expression in primary human hepatocytes. Figure 12D shows a correlation study using Child-Pugh score, HNF4α, and POM121C protein expression, performed using simple linear regression. Black circles refer to controls, light gray circles refer to child B, and dark gray circles refer to child C (R2 = 0.06, p = 0.23, n = 25). [Figure 13-1] Figures 13A-13D show SREBP1 expression in primary human hepatocytes. Figure 13A shows that primary human hepatocytes isolated from livers of patients undergoing liver transplantation for NASH or alcohol-induced cirrhosis were analyzed by Western blot for the expression of SREBP1 (Abcam ab28481 1:500) and HNF4α (ab41898 1:1000). [Figure 13-2]Figures 13A-13D show SREBP1 expression in primary human hepatocytes. Figures 13B and 13C show the relative intensities of SREBP1 among control, child B, and child C hepatocytes, compared by Brown-Forsythe one-way ANOVA and Welch's ANOVA test for multiple comparisons. Figure 13B: n=25. Figure 13C: R2=0.02, p=0.54, n=19. [Figure 13-3] Figures 13A-13D show SREBP1 expression in primary human hepatocytes. Figure 13D shows a correlation study using Child-Pugh score, HNF4α, and SREBP1 protein expression, performed using simple linear regression. Black circles refer to controls, light gray circles refer to child B, and dark gray circles refer to child C (R2 = 0.01, p = 0.86, n = 25). [Figure 14-1] Figures 14A-14D show EP300 expression in primary human hepatocytes. Figure 14A shows that primary human hepatocytes isolated from livers of patients undergoing liver transplantation for NASH or alcohol-induced cirrhosis were analyzed by Western blot for the expression of EP300 (Novus NB100-616 1:500) and HNF4α (ab41898 1:1000). [Figure 14-2] Figures 14A-14D show EP300 expression in primary human hepatocytes. Figures 14B and 14C show the relative intensities of EP300 among control, child B, and child C hepatocytes, compared by Brown-Forsythe one-way ANOVA and Welch's ANOVA test for multiple comparisons. Figure 14B: n=25. Figure 14C: R2=0.32, p=0.01, n=19. [Figure 14-3] Figures 14A-14D show EP300 expression in primary human hepatocytes. Figure 14D shows a correlation study using Child-Pugh score, HNF4α, and EP300 protein expression, performed using simple linear regression. Black circles refer to controls, light gray circles refer to child B, and dark gray circles refer to child C (R2 = 0.01, p = 0.69, n = 25). [Figure 15]Figures 15A and B show that CRISPR / Cas9 knockout of EP300, MTF1, NR0B2, NR5A2, POM121C, PROX1, or SREBP1 was performed in HepG2 cells, and the subcellular localization of HNF4α was analyzed by immunofluorescence (ab41898 1:500). The total number of DAPI- and HNF4α-positive nuclei (Figure 15A) and cytoplasmic HNF4α-positive cells (Figure 15B) was counted. Statistical analysis was performed using Brown-Forsythe one-way ANOVA and Welch's ANOVA test for multiple comparisons. Knockout of EP300, MTF1, NR0B2, NR5A2, POM121C, PROX1, and SREBP1 showed reduced nuclear localization of HNF4α and increased cytoplasmic localization of HNF4α. *p<0.05. [Figure 16] Figure 16 shows that primary human hepatocytes isolated from patients with NASH who underwent liver transplantation were transduced with AAV-HNF4α and AAV-MTF1, NR0B2, NR5A2, POM121C, PROX1, SREBP1, or GFP at an MOI of 10. The percentage of HNF4α-positive nuclei was counted. Statistical analysis was performed using Brown-Forsythe one-way ANOVA and Welch's ANOVA test for multiple comparisons. Co-transduction with HNF4α, MTF1, NR0B2, POM121C, PROX1, and SREBP1 resulted in an increase in the number of positive nuclei compared to the GFP-HNF4α co-transduction group (****p<0.0001, ***p<0.0005, **p<0.001, *p<0.05). DETAILED DESCRIPTION OF THE INVENTION

[0020] As used herein, the expression of the transcription factor HNF4α in the nuclei of hepatocytes in a subject and / or Compositions for treating liver disease in a subject by increasing transport or retention and methods are disclosed. In some embodiments, the methods involve detecting PROX1, NR 5A2, NR0B2, MTF1, SREBP1, EP300, and POM121C, etc. and functional fragments thereof. Upregulating the function and / or expression of one or more transcription factors AJB1 / HSP40, ATF6, ATF4, and PERK, and their functional fragments These transcription factors downregulate the expression or function of HNF4α. and modulating the expression and / or localization of It is an amazing discovery that this can be done.

[0021] In some embodiments, the method comprises administering a vector, the vector comprising: PROX1, NR5A2, NR0B2, MTF1, SREBP1, EP300, and P OM121C, and functional fragments thereof. It includes nucleic acids (e.g., DNA, ceDNA, or mRNA) encoding the factors. In some embodiments, the method comprises generating a gene encoding HNF4α (e.g., HNF4α isoform 2). by administering a vector containing a nucleic acid (e.g., DNA, ceDNA, or mRNA) that encodes the In other or further embodiments, the method comprises administering the composition. and wherein the composition comprises DNAJB1 / HSP40, ATF6, ATF4, and PERK. and functional fragments thereof. In another embodiment, the method reduces or inhibits the function of Lys106. Increased acetylation of HNF4α in the brain, increased expression of cMET, and This includes increasing activation of AKT via phosphorylation at Thr308 and / or Thr308. The method disclosed herein surprisingly increases the amount of HNF4α in the nuclei of hepatocytes. Such manipulation of HNF4α may improve hepatocyte function in patients with liver disease. Improve performance.

[0022] Terms used throughout this application shall be interpreted with the ordinary and typical meaning to those skilled in the art. However, applicants are encouraged to give specific definitions to the following terms: I hope.

[0023] term As used in this specification and claims, the singular forms "a," "an," and "The" includes plural references unless the context clearly indicates otherwise. Examples: For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

[0024] As used herein, "about" refers to a measurable value, e.g., an amount, a percentage, etc. The term "variation" refers to a variation of ±20%, ±10%, ±5%, or ±1% from the measurable value. It means to include.

[0025] "Administration" or "administering" to a subject includes any act of introducing or delivering an agent to a subject. Administration can be by any suitable route, including intravenous, intraperitoneal, etc. Administration includes self-administration and the administration by another.

[0026] As used herein, the term "comprising" and its variations includes is used interchangeably with the term "including" and variations thereof and is open The term "comprises" is a non-limiting term. Although we have used the terms "single" and "including," we do not mean "essentially consisting of" The terms "consisting essentially of" and "consisting of" Used instead of "comprising" and "including" to be more specific Embodiments can be provided and are disclosed.

[0027] "Composition" refers to any agent that has a beneficial biological effect. These include therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and preventative effects. , for example, to prevent injury or other undesirable physiological conditions (e.g., liver disease) The term also includes, but is not limited to, vectors, polynucleotides, cells, and the like. Cells, salts, esters, amides, proagents, active metabolites, isomers, fragments , analogs, and the like of the beneficial agents specifically mentioned herein. It also includes pharmacologically active derivatives. or where a particular composition is specifically identified, the term includes the composition itself, as well as Pharmacologically active vectors, polynucleotides, salts, and esters that are pharmaceutically acceptable , amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc. In some embodiments, the compositions disclosed herein are understood to include contains vectors, and the vectors are PROX1, NR5A2, NR0B2, MTF1, SR From the group consisting of EBP1, EP300, and POM121C, and functional fragments thereof. In some embodiments, the nucleic acid encoding one or more selected transcription factors is The compositions disclosed herein contain DNAJB1 / HSP40, ATF6, ATF4, and and PERK, and functional fragments thereof. In some embodiments, the nucleic acid comprises a nucleic acid that reduces the amount of or inhibits the function of a factor. The compositions disclosed herein include a vector, wherein the vector encodes HNFα. Contains nucleic acids.

[0028] "Effective amount" includes, but is not limited to, an amount that alleviates symptoms of a disease state or disorder (e.g., liver disease). The term "antibody" encompasses an amount capable of ameliorating, reversing, alleviating, preventing, or diagnosing a condition or symptom thereof. Unless otherwise indicated, explicitly or by context, an "effective amount" is defined as an amount sufficient to improve a condition. The minimum amount is not limited to the severity of the disease or disorder, and the effectiveness of the prevention and treatment of the disease or disorder. The ability of a treatment to reduce or alleviate the risk of a disease may be determined, without limitation, by biomarkers. or clinical parameters. The term "effective amount of a composition" refers to an amount of a composition that causes some relief of liver disease or restoration of liver function. It refers to an amount of a vector or composition sufficient to

[0029] A "fragment" is a peptide whose activity is comparable to that of the unmodified peptide, whether or not it is linked to other sequences. As long as it is not significantly altered or impaired compared to the insertion, deletion, substitution, or other selected modification of regions or specific amino acid residues. These modifications can be, for example, the removal or addition of amino acids capable of disulfide bonding. Adding nutrients to the organism, increasing its bio-longevity, and modifying its secretory properties In either case, the fragment may be provided with some additional properties, such as It is necessary for the compound to have a biologically active property such as regulating the transcription of a target gene.

[0030] The term "gene" or "gene sequence" refers to a coding or regulatory sequence, or A gene refers to any combination of coding and regulatory sequences, or fragments thereof. Thus, a "gene" as referred to herein may include a naturally occurring gene. A polynucleotide sequence as referred to herein may be all or part of a sequence. It may be used interchangeably with the term "gene" or any coding sequence, non-coding "Gene" or "genes" may include sequences or regulatory sequences, fragments thereof, and combinations thereof. The term "gene sequence" refers to, for example, a regulatory sequence upstream of a coding sequence (e.g., a ribosomal sequence). It contains the ribosomal binding site.

[0031] "Liver disease" as used herein generally refers to any disease, disorder, or condition that affects the liver. and conditions, such as the simple accumulation of fat in liver cells (steatosis), non-alcoholic Nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), alcoholic Liver disease (ALD), alcohol-related liver disease (including but not limited to fatty liver, alcohol alcohol-related hepatitis, including alcohol-related cirrhosis, macrovesicular steatosis is), periportal and lobular inflammation (steatohepatitis), cirrhosis, fibrosis, hepatic ischemia, hepatocellular carcinoma liver cancer, including hepatitis A, hepatitis B, hepatitis C, idiopathic liver disease, end-stage liver disease, and liver "Cirrhosis" as used herein refers to the progression of liver tissue damage and can have a wide range of severity, including liver failure. Hepatic cirrhosis is defined as a chronic disease of the liver characterized by fibrous thickening and / or regenerative nodules. The degree or severity of the "disease" is indicated by the Child-Pugh score. The classification can be divided into five clinical items: total bilirubin, serum albumin, The levels of prothrombin time prolongation, ascites, and hepatic encephalopathy were associated with various levels of each clinical item. The scores are calculated using a scoring system with values of 1, 2, and 3 corresponding to each item. A score of 3 is assigned to the most severe cases. The total scores for all five items are added together to determine the severity of the condition. Child-Pugh score and classification A score of 5-6 is designated Child-Pugh Class A, and a score of 7-9 is designated Child-Pugh Class B. A score of 10-15 is designated as Child-Pugh Class B, and a score of 10-15 is designated as Child-Pugh Class C. In general, Child-Pugh class A indicates the least severe liver disease, and Child-Pugh class B indicates the least severe liver disease. Pugh class C indicates the most severe liver disease. Therefore, some embodiments In accordance with the present disclosure, the methods are directed to treating Child-Pugh class B liver disease or Can be used to treat subjects with Child-Pugh class C liver disease In some embodiments, the methods disclosed herein include Child-Pugh's It can be used to treat subjects with liver disease. The method improves the subject's Child-Pugh score. In some embodiments, the method disclosed herein is This method can be used to treat ischemic donor livers by ex vivo perfusion. The invention provides a method for treating liver cancer before or after cancer treatment, including before or after liver resection. It can be used to

[0032] The term "nucleic acid" as used herein refers to a nucleic acid that is a nucleotide, e.g., a deoxyribonucleic acid. Polymers composed of dinucleotides (DNA) or ribonucleotides (RNA) The terms "ribonucleic acid" and "RNA" as used herein mean As used herein, "deoxynucleotide" refers to a polymer composed of polynucleotides. The terms "deoxyribonucleic acid" and "DNA" refer to nucleic acids composed of deoxyribonucleotides. In some embodiments, nucleic acid refers to a polymer of DNA (e.g., ceDNA or In some embodiments, the nucleic acid is mRNA.

[0033] The term "polynucleotide" refers to a single-stranded polymer composed of nucleotide monomers. refers to a mer or double-stranded polymer.

[0034] The term "polypeptide" refers to a single chain of D- or L-amino acids, or It consists of a mixture of D- and L-amino acids linked by peptide bonds. It refers to a compound that can be

[0035] The term "promoter" or "regulatory element" refers to a promoter or a regulatory element upstream or downstream from the initiation of transcription. Located downstream, it is involved in the recognition and transcription of RNA polymerase and other proteins to initiate transcription. The promoter must be of bacterial origin. For example, promoters derived from viruses or other organisms may be used in the compositions described herein. The present invention may be used in a composition, system, or method.

[0036] A "pharmaceutically acceptable carrier" (sometimes called a "carrier") is generally a safe, non-toxic means a carrier or excipient useful for preparing a pharmaceutical or therapeutic composition for animal use; "Carrier" or "carrier" includes carriers acceptable for pharmaceutical or therapeutic use in humans and / or humans. The term "pharmaceutically acceptable carrier" includes, but is not limited to, phosphate buffered saline. Saline solutions, water, emulsions (e.g., oil / water or water / oil emulsions) and / or or various types of wetting agents.

[0037] As used herein, the term "carrier" refers to any excipient, diluent, filler, Salts, buffers, stabilizers, solubilizers, lipids, stabilizing agents, or for use in pharmaceutical formulations The selection of a carrier for use in the composition will depend on its Pharmaceutically acceptable carriers containing these materials depend on the intended route of administration of the composition. and preparation of formulations are described, for example, in Remington's Pharmaceutical Sciences, 21st Edition, e.g. d. University of the Sciences in Philadelphia, Lippincott, Williams & Wilkins, P Hiladelphia, PA, 2005. Examples of physiologically acceptable carriers include saline. Saline, glycerol, DMSO, buffers such as phosphate buffer, citrate buffer, and others buffers containing organic acids; antioxidants containing ascorbic acid; low molecular weight (less than about 10 residues) poly(ethylene glycol) peptides; proteins, e.g., serum albumin, gelatin, immunoglobulins; hydrophilic polymers, e.g., polyvinylpyrrolidone; amino acids, e.g., glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and glucose, mannosaccharides sugars, or other carbohydrates, including dextrins; chelating agents, e.g., EDTA; sugar alcohols Cholesterol, e.g., mannitol or sorbitol; a salt-forming counterion such as sodium and / or non-ionic surfactants, such as TWEEN™ (ICI, In c.; Bridgewater, New Jersey), polyethylene glycol (P EG), and PLURONICS™ (BASF; Florham Park, NM J) to provide for administration of such a dose to provide the desired therapeutic treatment. The compositions disclosed herein advantageously comprise a weight percent of the total composition, including the carrier or diluent. Based on the amount, the total amount of one or more target compounds is about 0.1% to 99% by weight. It is possible.

[0038] The term "subject," as used herein, includes, but is not limited to, primates (e.g., humans) mammals, including cattle, sheep, goats, horses, dogs, cats, rabbits, rats, mice, etc. In some embodiments, the subject is a human. .

[0039] As used herein, the term "transcription factor" refers to a molecule that mediates the process of transcribing DNA into RNA. Typically, transcription factors are proteins that regulate the promoter or Transcription factors also have domains that bind to RNA polymerases or enhancer regions. It has domains that interact with enzymes and / or several other transcription factors, and This interaction can regulate the amount of RNA that is ultimately transcribed from DNA. The factor is present in the cytoplasm and can translocate to the nucleus upon activation.

[0040] As used herein, "treat," "treating," " The term "treatment" and its grammatical variants refer to one or more of the disorders or conditions. Partially or completely delaying, alleviating, mitigating or reducing the intensity of several associated symptoms; and / or alleviate, mitigate, or inhibit one or more causes of the disorder or condition. Treatment according to the present invention may be prophylactic, prophylactically, palliative or Treatment can be administered therapeutically. Treatment can be pre-symptomatic (e.g., before overt signs of liver disease), During early onset (e.g., at the first signs and symptoms of liver disease), the onset of liver disease was established Preventive treatment is administered to subjects after liver disease symptoms appear. It can take place over a period of days to years.

[0041] In some cases, "treat," "treating," or "treatment" The term "treatment" and its grammatical variants refer to the condition of a subject compared to before treatment or in the general population. and the incidence of liver disease in subjects compared with the incidence of such conditions in a group or study population. to reduce liver damage, restore liver function, and / or increase the amount of HNFα in the nuclei of hepatocytes. This includes adding

[0042] As used herein, a "vector" refers to a vector that contains an isolated nucleic acid. A vector is a composition of matter that can be used to deliver a vector to the interior of a cell. They are either self-replicating extrachromosomal vectors or vectors that integrate into the host genome. Alternatively, the vector may also be a vehicle that contains the aforementioned nucleic acid sequence. Vectors include plasmids, bacteriophages, and viral vectors (isolated, attenuated, (e.g., encapsulated, recombinant, encapsulated as virus particles), liposomes, exosomes The vector may be a double-stranded vector, such as a vesicle, extracellular vesicle, microparticle, and / or nanoparticle. or single-stranded DNA, RNA, or double- and / or single-stranded nucleotides In some embodiments, the vector may comprise a hybrid DNA / RNA sequence comprising packaging one or more nucleic acid sequences encoding one or more polypeptides a viral vector containing a nucleic acid sequence that is a viral packaging sequence responsible for In some embodiments, the vector is a plasmid. In some embodiments, the vector is an exosome. In some embodiments, the vector is a viral particle. In some embodiments, the viral particle is a lentiviral particle. In embodiments, the vectors are viruses with natural and / or engineered capsids. In some embodiments, the vector is operably linked to a regulatory sequence. and a viral particle comprising a nucleic acid sequence selected from the group consisting of one or more AAV viruses. Some embodiments encode fusion proteins containing the rRNA particle polypeptide or a fragment thereof. In embodiments, the vector is a nanoparticle comprising a nucleic acid or a polypeptide. In one embodiment, the vector is a lipid-based nanoparticle.

[0043] composition As described above, the present invention relates to the expression of the transcription factor HNF4α in the nuclei of hepatocytes in a subject. and / or transport and / or retention in the liver, thereby treating liver disease. In some embodiments, the method comprises treating a patient with a protease inhibitor, such as PROX1, NR2, or NR3. from 5A2, NR0B2, MTF1, SREBP1, EP300, and POM121C upregulating the expression or function of one or more transcription factors selected from the group consisting of and / or DNAJB1 / HSP40, ATF6, ATF4, and PERK In some embodiments, the method includes regulating a cell (e.g., a liver and optionally increasing the expression of endogenous HNFα within the nucleus of the cell, preferably within the nucleus of the cell. Expression of HNF4α was increased by adding exogenous HNFα or by introducing exogenous HNFα. As used herein, the term "PROX1" refers to a protein that is involved in the expression or function of a protein. NR5A2, NR0B2, MTF1, SREBP1, EP300, and POM121C Upregulation of and / or DNAJB1 / HSP40, ATF6, A Downregulation of TF4 and PERK significantly increased the level of HNF4α in the nucleus of hepatocytes. It has been described that this increases liver function, leading to the restoration of liver function and the alleviation of liver disease. In one embodiment, the method increases the acetylation of HNF4α at Lys106, By increasing the expression of MET and / or AK via phosphorylation at Thr308 These methods include increasing the activation of T. In combination with the method described in U.S. Patent Application Publication No. 2014 / 0249209, They can be used in combination.

[0044] In some embodiments, the method comprises detecting a gene encoding a nucleotide sequence ... 1, SREBP1, EP300, and POM121C. or multiple transcription factors, upregulating the expression or function of HNF4α. In some embodiments, the composition further comprises: In some embodiments, this further comprises upregulating the expression or function of a The method includes upregulating the expression or function of HNF4α and SREBP1. In some embodiments, the method further comprises: and upregulating the expression or function of SREBP1.

[0045] Therefore, the present specification includes PROX1, NR5A2, NR0B2, MTF1, SRE One or more selected from the group consisting of BP1, EP300, and POM121C Increases the expression or function of transcription factors and / or DNAJB1 / HSP40, one or more transcription factors selected from the group consisting of ATF6, ATF4, and PERK In some embodiments, the composition comprises a compound that reduces the expression or function of a gene. , PROX1, NR5A2, NR0B2, MTF1, SREBP1, EP300, and POM121C, HNF4, and one or more transcription factors selected from the group consisting of In some embodiments, the composition upregulates the expression or function of H. Upregulating the expression or function of NF4α and PROX1. In one embodiment, the composition upregulates the expression or function of HNF4α and SREBP1. In some embodiments, the composition comprises HNF4α, PROX1, and SRE. Upregulating BP1 expression or function.

[0046] The present specification provides a composition comprising a vector, wherein the vector is a vector encoding PROX1, NR5A2 , NR0B2, MTF1, SREBP1, EP300, and / or POM121C, and functional fragments thereof. In some embodiments, the vector comprises a nucleic acid encoding a PRO In another embodiment, the vector comprises a nucleic acid encoding X1 or a functional fragment thereof. In another embodiment, the vector comprises a nucleic acid encoding R5A2 or a functional fragment thereof. In another embodiment, the vector comprises a nucleic acid encoding NR0B2 or a functional fragment thereof. In another embodiment, the vector comprises a nucleic acid encoding MTF1 or a functional fragment thereof. The target comprises a nucleic acid encoding SREBP1 or a functional fragment thereof. The vector comprises a nucleic acid encoding EP300 or a functional fragment thereof. In some embodiments, the vector comprises a nucleic acid encoding POM121C or a functional fragment thereof. In some embodiments, the vector further comprises a nucleic acid encoding HNF4α. In this embodiment, the vector comprises nucleic acids encoding PROX1 and SREBP1. In some embodiments, the vector encodes HNF4α, PROX1, and SREBP1. In some embodiments, the vector comprises a nucleic acid encoding HNF4α and PRO. In some embodiments, the vector comprises a nucleic acid encoding HNF4α and X1. It contains a nucleic acid encoding SREBP1.

[0047] In addition to the liver, HNF4α is also highly expressed in the kidney, small intestine, colon, and pancreas. Polymorphic mutations in the HNF4α gene are associated with early-onset adult associated with a wide range of diseases, including type 2 diabetes (MODY), Crohn's disease, and inflammatory bowel syndrome Transcription from the P1 or P2 promoter combined with alternative splicing can generate 12 different transcripts. Relative isoform expression is determined by the The 12 isoforms are responsible for transcriptional activation and repression, respectively. , which differ only at the N- and C-termini (see, e.g., the entirety of which is incorporated herein by reference). (See Ko et al., Cell Rep. 2019 Mar 5; 26(10):2549-2557.e3). These forms play distinct functions that regulate specific subsets of genes in a tissue-dependent manner. For example, HNF4α isoform 2 has been reported. is abundant in the liver and acts as a tumor suppressor, and its loss is described in this application HNF4α isoform 8 is associated with hepatocellular carcinoma or liver failure in the colon, whereas HNF4α isoform 8 is associated with hepatocellular carcinoma or liver failure in the colon. It is highly expressed and regulates the expression of growth-promoting genes.

[0048] Thus, in some embodiments, the vectors disclosed herein are capable of expressing HNF Further, the nucleic acid encoding the 4α isoform 2 polypeptide is included. In the present invention, the HNF4α isoform 2 polypeptide has at least about 80% identity with SEQ ID NO: 1, Containing about 85%, about 90%, about 95%, or about 98% identical sequences or fragments thereof. In some embodiments, the nucleic acid has at least about 80%, about 85%, or about the same sequence as SEQ ID NO:31. 90%, about 95%, or about 98% identical to, or a fragment thereof. In the mouse model, HNF4α isoform 2 polypeptide is driven by promoter 1 (P1), In other words, its expression is driven by the P1 promoter of HNF4α. Herein, it is referred to as HNF4α isoform 2 (P1). In embodiments, the HNF4α isoform 2 polynucleotide or nucleic acid comprises a P1 promoter. The motor is operably connected to the motor.

[0049] A vector can be a nucleic acid sequence that contains regulatory nucleic acid sequences that control the replication of expressible genes. In some embodiments, a promoter (e.g., vectors containing polynucleotides encoding transcription factors (e.g., polynucleotides encoding transcription factors) are the result of human manipulation. (e.g., Sambrook et al., Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999) ng Harbor Laboratory, Cold Spring Harbor, NY, (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998) a second nucleic acid (e.g., a polynucleotide encoding a transcription factor) Any promoter described herein may be included that is heterologous to the promoter. The vector of this embodiment can be operably linked to one or more of the transcription factors described above. , promoters, enhancers, antibiotic resistance genes, and / or initiation sites. It is to be understood herein that the present invention may include the following:

[0050] In some embodiments, the vector may be a viral vector. "Viral vector" when used in reference to a vehicle includes a virus, virus-like particle, or Packaging of nucleic acid sequences in a virus, virion, viral particle, or pseudotyped virus Any virus, virus-like particle, virion, virus containing a nucleic acid sequence that directs binding. In some embodiments, the virus A virus-like particle, a virion, a virus particle, or a pseudotyped virus is a vector. - (e.g., nucleic acid vectors) into and / or between host cells In some embodiments, viruses, virus-like particles, virions, viral particles, The vector, or pseudotyped virus, delivers a vector (e.g., a nucleic acid vector) to the liver of a subject. They can be transported into and / or between target cells, such as liver cells in the liver. Importantly, in some embodiments, viruses, virus-like particles, virions, viruses The pseudovirus, or pseudotyped virus, transports to the nucleus of target cells (e.g., hepatocytes). The term "viral vector" may also be used as a reference for any purpose. No. 2018 / 0057839, the disclosure of which is incorporated herein by reference. It is intended to refer to those forms that are fully described. Suitable viral vectors include: For example, adenovirus, adeno-associated virus (AAV), vaccinia virus, hepatitis B virus, pesviruses, baculoviruses and retroviruses, parvoviruses, and lentiviruses In some embodiments, the viral vector is a lentiviral vector. vector or adeno-associated virus vector.

[0051] The construction of replication-deficient adenoviruses has been described (Berkner et al., J. Virology 61:1 213-1220 (1987);Massie et al., Mol. Cell. Biol. 6:2872-2883 (1986);Haj-Ahmad e t al., J. Virology 57:267-274 (1986);Davidson et al., J. Virology 61:1226-1239 (1987) ;Zhang "Generation and identification of recombinant adenovirus by lipos "ome-mediated transfection and PCR analysis" BioTechniques 15:868-872 (1993)). The advantage of using these viruses as vectors is that they cannot replicate within the initially infected cells. can produce new infectious virus particles but cannot spread to other cell types The potential scope of recombinant adenoviruses is limited. after in vivo delivery directly to vascular endothelium, CNS parenchyma, and many other tissue sites. It has been shown that highly efficient gene transfer can be achieved (Morsy, J. Clin. Invest. 92:1 580-1586 (1993);Kirshenbaum, J. Clin. Invest. 92:381-387 (1993);Roessler, J.C lin. Invest. 92:1085-1092 (1993);Moullier, Nature Genetics 4:154-159 (1993);La Salle, Science 259:988-990 (1993); Gomez-Foix, J. Biol. Chem. 267:25129-25134 ( 1992);Rich, Human Gene Therapy 4:461-476 (1993);Zabner, Nature Genetics 6:75-8 3 (1994);Guzman, Circulation Research 73:1201-1207 (1993);Bout, Human Gene The rapy 5:3-10 (1994);Zabner, Cell 75:207-216 (1993);Caillaud, Eur. J. Neuroscien ce 5:1287-1291 (1993); and Ragot, J. Gen. Virology 74:501-507 (1993)). Adenoviruses achieve gene transfer by binding to specific cell surface receptors, The virus then undergoes receptor-mediated replication, similar to wild-type or replication-deficient adenovirus. It is internalized by endocytosis (Chardonnet and Dales, Virology 40:462-477 (1970);Brown and Burlingham, J. Virology 12:386-396 (1973);Svensson and Persso n, J. Virology 55:442-449 (1985);Seth, et al., J. Virol. 51:650-655 (1984);Set h, et al., Mol. Cell. Biol. 4:1528-1533 (1984);Varga et al., J. Virology 65:606 1-6070 (1991); Wickham et al., Cell 73:309-319 (1993)).

[0052] Another type of viral vector is based on adeno-associated virus (AAV). This defective parvovirus can infect many cell types and is non-pathogenic to humans. V-type vectors can transport approximately 4–5 kb, and wild-type AAV is stably attached to chromosome 19. AAV inverted terminal repeats (ITRs) or modifications thereof are known to selectively insert Conferring site-specific integration and cytotoxicity, but not cytotoxicity, the promoter provides cell-specific expression Reference is made to U.S. Patent No. 6,261,834 for reference to AAV vectors. The specification is incorporated herein by reference. Methods for using AAV vectors for this purpose are known in the art. See U.S. Patent No. 9,981,048, which is incorporated herein by reference.

[0053] Viral vectors, particularly adenoviral vectors, are often mediated by cationic amphiphiles, e.g. , cationic lipid, poly-L-lysine (PLL), and diethylaminoethyl dextran It can form a complex with a virus (DELAE-dextran) and other compounds, allowing it to penetrate into target cells. Virus infection efficiency is improved (see, e.g., 1997, 1 (See PCT / US97 / 21496, filed January 20th.) See, for example, Zhong et al., J. Genet Syndrome Gen., the disclosure of which is incorporated herein. e Therapy 2012 Jan. 10; S1. pii: 008, U.S. Patent No. 5,139,941, U.S. Patent No. Nos. 5,252,479 and 5,753,500, and PCT application Ser. Nos. AAV vectors such as those disclosed in published International Publication No. WO 97 / 09441 are also known. The target also shows that these vectors integrate into the host chromosome, allowing for repeated administration of the vector. Viral vectors are useful because they minimize the need for transfection. For details, see McConnell et al., 2004, Hum Gene Ther. 15(11):1022-33; McCarty et al. ., 2004, Annu Rev Genet. 38:819-45;Mah et al., 2002, Clin. Pharmacokinet. 41(12 ):901-11; Scott et al., 2002, Neuromuscul. Disord. 12(Suppl 1):S23-9 stomach.

[0054] In some embodiments, the vector is a nanoparticle. Nanoparticles as used herein As used herein, a nanoparticle can be any nanoparticle useful for delivering nucleic acids. The term "nanoparticles" refers to the nanoparticles that are present in sufficient numbers to be effective after delivery to the application or treatment site. chemical and / or physical resistance to the environment of such use so as to remain substantially intact They are biocompatible, sufficiently resistant to physical destruction, and have a size in the nanometer range. In some embodiments, nanoparticles refer to lipid-like nanoparticles. See, for example, International Publication No. WO 2016 / 187531, which is incorporated herein by reference. No. Brochure, International Publication No. 2017 / 176974 Brochure, International Publication No. 201 9 / 027999 Brochure, or Li, B et al., An Orthogonal array optimiza tion of lipid-like nanoparticles for mRNA delivery in vivo. Nano Lett. 2015, 15, In some embodiments, the nanoparticles are comprised of a lipid bilayer or a lipid nanoparticle. In some embodiments, the vector is an mRNA lipid nanoparticle. do.

[0055] In some embodiments, the disclosed nanoparticles can be used to target biological entities, e.g., target cells. specific membrane components or cell surface receptors (e.g., receptors or receptors that facilitate delivery to hepatocytes) receptors on hepatocytes) or otherwise associate with it. The disclosed nanoparticles bind to receptors (e.g., , hepatic asialoglycoprotein receptor (ASGPR) or low-density lipoprotein (LDL) The nucleotide sequence can be engineered to contain a ligand that binds to a receptor (R).

[0056] In some aspects, the nanoparticles disclosed herein are capable of delivering nucleic acids to hepatocytes. The nanoparticles may contain supplemental ingredients that promote the release of cholesterol. In some embodiments, the polymer may include hydroxybenzoates, ... The nanoparticles were 5A2-SC8, 1,2-dioleoyl-sn-glycero-3-phosphoeta diolamine (DOPE), cholesterol, and / or 1,2-dimyristoyl- rac-glycerol-methoxy(poly(ethylene glycol)), or any of these In some embodiments, the nanoparticles comprise a combination of 5A2-SC8, 1,2-dioxanediol, Dodecyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol , and 1,2-dimyristoyl-rac-glycerol-methoxy(poly(ethylene glycol) In some embodiments, the nanoparticles further comprise 1,2-dioleoyl Some examples further include dibenzo-3-trimethylammonium-propane (DOTAP). In an embodiment, the nanoparticles 5A2-SC8, 1,2-dioleoyl-sn-glycero-3- Phosphoethanolamine (DOPE), cholesterol, and 1,2-dimyristoyl The molar ratio of -rac-glycerol-methoxy(poly(ethylene glycol)) is approximately 15 / 15 / 30 / 3.

[0057] In some embodiments, the nanoparticles comprise DLin-MC3-DMA, 1,2-distearate diisopropyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1, 2-Dimyristoyl-rac-glycerol-methoxy(poly(ethylene glycol)) In some embodiments, DLin-MC3-DMA, 1,2-distearoyl -sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1,2-diphosphoric acid Molar ratio of myristoyl-rac-glycerol-methoxy(poly(ethylene glycol)) The ratio is approximately 50 / 10 / 38.5 / 1.5.

[0058] In some embodiments, the nanoparticles are C12-200, 1,2-dioleoyl-sn -glycero-3-phosphoethanolamine (DOPE), cholesterol, and 1,2 -Dimyristoyl-rac-glycerol-methoxy(poly(ethylene glycol)) In some embodiments, C12-200, 1,2-dioleoyl-sn-glycerol D-3-phosphoethanolamine (DOPE), cholesterol, and 1,2-dimethicone The molar ratio of stoyl-rac-glycerol-methoxy(poly(ethylene glycol)) is , approximately 35 / 16 / 46.5 / 2.5.

[0059] In some embodiments, the nanoparticles disclosed herein include 5A2-SC8, 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), Sterol, 1,2-dimyristoyl-rac-glycerol-methoxy(poly(ethylene glycol), and 1,2-dioleoyl-3-trimethylammonium-propane The nanoparticles contain about 0.1% to about 30% mol / mol of DOTAP. For example, the amount of DOTAP present in the nanoparticles may vary depending on the nanoparticle size. Approximately 0.1%, approximately 0.2%, approximately 0.3%, approximately 0.4%, approximately 0.5%, approximately 0.6% mol / mol, about 0.7%mol / mol, about 0.8%mol / mol, about 0.9%mol / m ol, about 1%mol / mol, about 2%mol / mol, about 2.5%mol / mol, about 3 %mol / mol, about 3.5%mol / mol, about 4%mol / mol, about 4.5%mo l / mol, about 5%mol / mol, about 5.5%mol / mol, about 6%mol / mol , about 6.5%mol / mol, about 7%mol / mol, about 7.5%mol / mol, about 8 %mol / mol, about 8.5%mol / mol, about 9%mol / mol, about 9.5%mo l / mol, about 10%mol / mol, about 10.5%mol / mol, about 11%mol / mol, about 11.5%mol / mol, about 12%mol / mol, about 12.5%mol / mol, about 13%mol / mol, about 13.5%mol / mol, about 14%mol / mol l, about 15%mol / mol, about 16%mol / mol, about 17%mol / mol, about 1 8%mol / mol, about 19%mol / mol, about 20%mol / mol, about 22%mo l / mol, about 24%mol / mol, about 26%mol / mol, about 28%mol / mol In some embodiments, the amount of hydroxybenzoates present in the nanoparticles may be about 30% mol / mol. The amount of DOTAP added is about 20% mol / mol of the nanoparticles.

[0060] In some embodiments, the nanoparticles disclosed herein for liver-specific delivery and methods are well known in the art, for example, in Cheng et al., Nat Nanotechnol. 2020 Apr; 15(4):313-320. Epub 2020 Apr 6;T repotec et al., Mol Ther. 2019 Apr 10; 27(4):794-802. Epub 2018 Dec 22;Truong, et al., Proc Natl Acad Sci USA. 2019 Oct 15; 116(42):21150-21159. Epub 2019 Sep It is described in 9.

[0061] In further embodiments, the vectors disclosed herein comprise poly(amido-amine ), poly-beta amino-ester (PBAE), and / or polyethyleneimine ( In some embodiments, the vector comprises polyacridine PEG. In some embodiments, the vectors disclosed herein comprise an outer PEG shell and a nanoparticle. It contains a nanoparticle-based core.

[0062] Lipid-based nanoparticles successfully deliver therapeutic payloads to the liver. For example, Witzig See mann et al., Adv Drug Deliv Rev. 2020 Jul, doi: 10.1016 / j.addr.2020.06.026 Liposomes can be prepared from several different types of lipids. However, phospholipids are the most commonly used to generate lipid-based nanoparticles as drug carriers. Lipid particles for use in the present invention comprise a liposome-forming lipid and a phospholipid. , as well as membrane active sterols (e.g., cholesterol). Liposomes may contain other lipids and phospholipids that are not liposome-forming lipids. can.

[0063] Phospholipids include, for example, lecithin (e.g., egg lecithin or soy lecithin); phosphatidylcholine (e.g., egg phosphatidylcholine); hydrogenated phosphatidylcholine; lysophosphatidylcholine Phosphatidylcholine; Dipalmitoylphosphatidylcholine; Distearoylphospha Phosphatidylcholine; Dimyristoyl phosphatidylcholine; Dilauroyl phosphatidylcholine glycerophospholipids (e.g., phosphatidylglycerol, phosphatidylserine, Phosphatidylethanolamine, Lysophosphatidylethanolamine, Phosphatidyl Inositol, phosphatidylinositol phosphate, phosphatidylinositol bisphosphate and phosphatidylinositol triphosphate); sphingomyelinositol Myelin; cardiolipin; phosphatidic acid; plasmalogen; or a mixture thereof Each possibility represents a separate embodiment of the present invention. Examples of other lipids that can be used include glycolipids (e.g., glyceroglycolipids, e.g., galactosyltransferases, etc.). glycosphingolipids and sulfolipids, glycosphingolipids, e.g., cerebrosides, glucocerebrosides and galactocerebrosides, and glycosylphosphatidylinositols); phospholipids (e.g., ceramide phosphorylcholine, ceramide phosphorylethanolamine, and and ceramide phosphorylglycerol); or mixtures thereof. The potential represents a separate embodiment of the present invention. Negatively or positively charged lipid nanoparticles can be For example, by using anionic or cationic phospholipids or lipids. Such anionic / cationic phospholipids or lipids are typically Lipophilic moieties such as sterols, acyl or diacyl chains, and the lipids are generally Taste has a negative / positive charge.

[0064] In some embodiments, the nanoparticles disclosed herein are one, two, three, or more biocompatible and / or biodegradable polymers. The nanoparticles are made of one or more polymers containing a biodegradable polymer and polyethylene glycol. About 10 to about 99 weight percent of a block copolymer and about 0 to about 99 weight percent of a biodegradable homopolymer. The polymer may comprise about 50 weight percent of the polymer. Degradable polymers include, for example, microcrystalline cellulose, hydroxypropyl cellulose, such as cellulose, hydroxypropyl methylcellulose, and polyethylene oxide (PEG). Polyalkylene oxides, polyanhydrides, poly(ester anhydrides), polyhydroxy acids, e.g. For example, polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) Polypropylene glycol acrylate (PLGA), poly-3-hydroxybutyrate (PHB) and their copolymers polymers, poly-4-hydroxybutyrate (P4HB) and their copolymers, poly Caprolactone and its copolymers, as well as combinations thereof, may be included.

[0065] In some embodiments, the nanoparticles have a diameter of about 1 nm to about 1000 nm. In some embodiments, the nanoparticles are, for example, about 1000 nm, about 950 nm, about 900 nm, nm, approx. 850nm, approx. 800nm, approx. 750nm, approx. 700nm, approx. 650nm, approx. 6 00nm, about 550nm, about 500nm, about 450nm, about 400nm, about 350nm, Approx. 300nm, approx. 290nm, approx. 280nm, approx. 270nm, approx. 260nm, approx. 250n m, approx. 240nm, approx. 230nm, approx. 220nm, approx. 210nm, approx. 200nm, approx. 19 0nm, approx. 180nm, approx. 170nm, approx. 160nm, approx. 150nm, approx. 140nm, approx. 130nm, approx. 120nm, approx. 110nm, approx. 100nm, approx. 90nm, approx. 80nm, approx. 70 nm, about 60 nm, about 50 nm, about 40 nm, about 30 nm, about 20 nm, or about 1 In some embodiments, the nanoparticles have a diameter of less than about 20 nm, for example. ~approx. 1000nm, approx. 20nm ~ approx. 800nm, approx. 20nm ~ approx. 700nm, approx. 30nm ~600nm, approx. 30nm ~ approx. 500nm, approx. 40nm ~ approx. 400nm, approx. 40nm ~ Approx. 300nm, approx. 40nm ~ approx. 250nm, approx. 50nm ~ approx. 250nm, approx. 50nm ~ approx. 200nm, about 50nm to about 150nm, about 60nm to about 150nm, about 70nm to about 1 50nm, about 80nm to about 150nm, about 90nm to about 150nm, about 100nm to about 1 50nm, about 110nm to about 150nm, about 120nm to about 150nm, about 90nm to about 140nm, about 90nm to about 130nm, about 90nm to about 120nm, 100nm to about 1 40nm, about 100nm~about 130nm, about 100nm~about 120nm, about 100nm~ Approx. 110nm, approx. 110nm ~ approx. 120nm, approx. 110nm ~ approx. 130nm, approx. 110n m ~ approx. 140nm, approx. 90nm ~ approx. 200nm, approx. 100nm ~ approx. 195nm, approx. 110 nm ~ approx. 190nm, approx. 120nm ~ approx. 185nm, approx. 130nm ~ approx. 180nm, approx. 1 40nm to approximately 175nm, 150nm to 175nm, or approximately 150nm to approximately 170nm In some embodiments, the nanoparticles have a diameter of about 100 nm to about 250 nm. In some embodiments, the nanoparticles have a diameter of about 150 nm to about 175 nm. In some embodiments, the nanoparticles have a diameter of about 135 nm to about 175 nm. The particles can have any shape, but are generally spherical.

[0066] In some embodiments, the vector used herein is an exosome. The terms "microvesicles" and "exosomes" as used herein refer to microvesicles of approximately 10 nm to 100 nm in size. About 5000 nm, more typically 30 nm to 1000 nm, most typically about 50 nm to Membrane particles with a diameter of 750 nm (or the largest dimension if the particles are not spherical) wherein at least a portion of the membrane of the exosome is obtained directly from a cell. Most commonly, exosomes have a size (average diameter) that is 5% or less of the size of the donor cells. Therefore, exosomes of particular interest include those excreted from cells. Methods for producing exosomes are known in the art. U.S. Patent Application Publication No. 2018 / 0177727, which is incorporated herein in its entirety. See, e.g., Exosomes and Immunoglobulins for Delivery of Polynucleotides and Polypeptides. and its uses are known in the art, and are incorporated herein by reference in their entirety. See U.S. Pat. No. 10,577,630.

[0067] Also described herein are methods for inhibiting the expression of PROX1, NR5A2, N through RNA activation (RNAa). From the group consisting of R0B2, MTF1, SREBP1, EP300, and POM121C Also included are compositions that increase the expression or function of one or more selected transcription factors. Thus, in some embodiments, the composition comprises: B2, MTF1, SREBP1, EP300, and POM121C It contains activating short hairpin RNA (shRNA).

[0068] "HNF4α" as used herein refers to the HNF4α gene encoded by the HNF4A gene in humans. In some embodiments, the HNF4α polypeptide refers to a polypeptide that is are identified in one or more publicly available databases such as :HGNC:5024, Entrez Gene:3172, Ensembl:ENSG 00000101076, OMIM:600281, UniProtKB:P41235 In some embodiments, the HNF4α polypeptide has the sequence of SEQ ID NO: 1 (HNF4α isoform 2), or about 80% or more, about 85% or more, about 90% or more of SEQ ID NO: 1; A polypeptide sequence having about 95% or more, or about 98% or more homology, or a sequence number The HNF4α polypeptide of SEQ ID NO: 1 includes a polypeptide containing a portion of the mature HNF4α polypeptide of SEQ ID NO: 1. It may represent the immature or preprocessed form of NF4α, and therefore, the sequence The mature or processed portion of the HNF4α polypeptide of SEQ ID NO: 1 is included. In this form, the HNF4α polypeptide is incorporated herein by reference for all purposes. The invention described in U.S. Patent Application Publication No. 2014 / 0249209 includes In some embodiments, the HNF4α polynucleotide is SEQ ID NO: 31, the sequence Sequence No. 32, SEQ ID NO: 33, or SEQ ID NO: 34, or SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34, about 80% or more, about 85% or more, about 90% or more or a polynucleotide sequence having about 95% or more, or about 98% or more homology thereto; is a polynucleotide containing a portion of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34. Contains nucleotides.

[0069] PROX1 (Prospero-related homeobox 1) is normally expressed in the mouse liver during liver organogenesis. It is first expressed in endoderm cells at embryonic day 8.5 (E8.5). In the adult liver, PROX The role of 1 may be to regulate the energy metabolism of hepatocytes. Prox1 binds to specific DNA elements by directly binding its homeodomain. It has been reported that PROX1 can function as an activator of gene transcription. The polypeptide encoded by the PROX1 gene in humans is herein In some embodiments, the PROX1 polypeptide is one or more of the following: is identified in multiple publicly available databases: HGNC:94 59, Entrez Gene:5629, Ensembl:ENSG00000117 707, OMIM:601546, UniProtKB:Q92786. Some implementations In one embodiment, the PROX1 polypeptide has the sequence of SEQ ID NO:2 or a sequence identical to or about 80% identical to SEQ ID NO:2. or more, about 85% or more, about 90% or more, about 95% or more, or about 98% or more homology or a polypeptide comprising a portion of SEQ ID NO: 2. The PROX1 polypeptide may represent an immature or preprocessed form of mature PROX1. Thus, the mature portion of the PROX1 polypeptide of SEQ ID NO: 2 or In some embodiments, the PROX1 polynucleotide comprises a processing moiety. SEQ ID NO: 13, or a sequence similar to SEQ ID NO: 13, but with a similar identity to ... but with a similar identity to SEQ ID NO: 13, Polynucleotide sequences with 5% or more, or about 98% or more homology, or sequence numbers The present invention also includes a polynucleotide comprising a portion of SEQ ID NO:13.

[0070] NR5A2 (nuclear receptor 5A2; liver receptor homolog-1; LRH-1) is one of the targets Binds as a monomer to specific response elements within gene promoters and regulatory regions NR5A2 is a nuclear receptor that also regulates bile acid synthesis enzymes, fatty acid metabolism, and mitochondria. "NR5A2" is a protein that can positively regulate genes encoding mitochondrial functions. In humans, it refers to the polypeptide encoded by the NR5A2 gene. In some embodiments, the NR5A2 polypeptide is derived from one or more publicly known It is identified in the database available at: HGNC:7984, Ent rez Gene:2494, Ensembl:ENSG00000116833, OM IM:604453, UniProtKB:O00482. In some embodiments, N The R5A2 polypeptide has a sequence similar to SEQ ID NO:3, or a sequence similar to SEQ ID NO:3, but not greater than about 80%, about 85%, or more. % or more, about 90% or more, about 95% or more, or about 98% or more homology The NR5A2 sequence of SEQ ID NO: 3 is a polypeptide containing a portion of the NR5A2 sequence of SEQ ID NO: 3. The polypeptide may represent an immature or preprocessed form of mature NR5A2, and therefore Thus, the mature or processed portion of the NR5A2 polypeptide of SEQ ID NO:3 is included herein. In some embodiments, the NR5A2 polynucleotide is SEQ ID NO: 14, SEQ ID NO: 5 15, or the sequence of SEQ ID NO: 16, or SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16 and about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about A polynucleotide sequence having 98% or more homology, or SEQ ID NO: 14, SEQ ID NO: 15 or a polynucleotide comprising a portion of SEQ ID NO:16.

[0071] NR0B2 (nuclear receptor small heterodimer partner; SHP) is normally expressed in normal It is highly expressed in various hepatocytes and is an important transcriptional regulator of bile acid, glucose, and lipid metabolism. SUMOylation of NR0B2 mediates nuclear transport and maintenance of bile acid homeostasis. and the role of SHP in feedback inhibition of bile acid biosynthesis, which is important for protection from hepatotoxicity. It may be required for gene repression (Kim DH et al., 2016). In humans, it refers to the polypeptide encoded by the NR0B2 gene. In some embodiments, the NR0B2 polypeptide is derived from one or more publicly known It is identified in the database available at: HGNC:7961, Ent rez Gene:8431, Ensembl:ENSG00000131910, OM IM:604630, UniProtKB:Q15466. In some embodiments, N The R0B2 polypeptide has a sequence similar to SEQ ID NO:4, or a sequence similar to SEQ ID NO:4, but which is about 80% or more similar to SEQ ID NO:4, % or more, about 90% or more, about 95% or more, or about 98% or more homology The polypeptide includes a polypeptide containing a portion of SEQ ID NO: 4. The polypeptide may represent an immature or preprocessed form of mature NR0B2, and therefore Thus, the mature or processed portion of the NR0B2 polypeptide of SEQ ID NO:4 is included herein. In some embodiments, the NR0B2 polynucleotide comprises the sequence of SEQ ID NO: 17. or about 80% or more, about 85% or more, about 90% or more, about 95% or more of SEQ ID NO: 17; or a polynucleotide sequence having about 98% or more homology thereto, or a portion of SEQ ID NO: 17 The polynucleotide comprises:

[0072] MTF1 (metal-responsive transcription factor 1) regulates basal transcription and heavy metal regulation of metallothionein genes. Other genes involved in cellular stress response and metal homeostasis can mediate both gene-induced transcription and MTF1 also regulates other metal-responsive genes, such as zinc transcription factors. MTF1 may also be involved in the transcriptional regulation of transporter 1. MTF1 regulates zinc levels in hepatocytes. "MTF1" as used herein refers to a signaling lymphocyte activation molecule. family of autoligand receptors, encoded in humans by the MTF1 gene In some embodiments, the MTF1 polypeptide refers to a polypeptide that is are identified in one or more publicly available databases such as :HGNC:7428, Entrez Gene:4520, Ensembl:ENSG 00000188786, OMIM:600172, UniProtKB:Q14872 In some embodiments, the MTF1 polypeptide has the sequence of SEQ ID NO: 5, or SEQ ID NO: No. 5 and approximately 80% or more, approximately 85% or more, approximately 90% or more, approximately 95% or more, or approximately 98% or more or a polypeptide comprising a portion of SEQ ID NO:5. The MTF1 polypeptide of SEQ ID NO: 5 represents an immature or preprocessed form of mature MTF1. The mature portion of the MTF1 polypeptide of SEQ ID NO: 5 is therefore referred to herein as the mature portion of the MTF1 polypeptide of SEQ ID NO: 5. In some embodiments, the MTF1 polynucleotide comprises: The sequence of SEQ ID NO: 18, or a sequence identical to SEQ ID NO: 18 by about 80% or more, about 85% or more, or about 90% or more , a polynucleotide sequence having about 95% or more, or about 98% or more homology thereto; It includes a polynucleotide comprising a portion of SEQ ID NO:18.

[0073] SREBP1 (sterol regulatory element-binding protein 1) is a cholesterol SREBP1 is a transcription factor involved in the biosynthesis of fatty acids, and triglycerides. It can regulate the expression and activity of the AKT / PI3K signaling pathway and vice versa It is also possible to synthesize SREBF1 (Shi Q et al., 2016; Porstmann T et al., 2008). , herein defined as an autoligand receptor for the signaling lymphocyte activation molecule family. It refers to the polypeptide encoded by the SREBF1 gene in humans. In some embodiments, the SREBF1 polypeptide is one or more of the following publicly known polypeptides: It is identified in publicly available databases as: HGNC:11289, E ntrez Gene:6720, Ensembl:ENSG00000072310, OMIM: 184756, UniProtKB: P36956. In some embodiments , the SREBF1 polypeptide has a sequence identical to SEQ ID NO: 6 or a sequence identical to SEQ ID NO: 6 by about 80% or more; A sequence having about 85% or more, about 90% or more, about 95% or more, or about 98% or more homology The polypeptide sequence of SEQ ID NO: 6 or a portion thereof is also included. The F1 polypeptide may represent an immature or preprocessed form of mature SREBF1, and Thus, the mature or processed portion of the SREBF1 polypeptide of SEQ ID NO:6 is referred to herein. In some embodiments, the SREBP1 polynucleotide comprises a portion of SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21, or SEQ ID NO: 19, SEQ ID NO: 20 or about 80% or more, about 85% or more, about 90% or more, about 95% or more of SEQ ID NO: 21; or a polynucleotide sequence having about 98% or more homology thereto, or SEQ ID NO: 19, It includes a polynucleotide comprising a portion of SEQ ID NO: 20 or SEQ ID NO: 21.

[0074] EP300 (histone acetyltransferase p300) EP300 is a C / EB Forms a complex with P protein and regulates triglyceride synthesis and glucose metabolism during the development of fatty liver and several transcription factors highly expressed in the liver, such as Foxo1 and Foxo2. Activating the promoter of genes involved in the regulation of farnesoid X receptor (FXR) "EP300" is used herein to refer to a member of the signaling lymphocyte activation molecule family. It is an autoligand receptor for leukemia, and in humans is encoded by the EP300 gene. In some embodiments, the EP300 polypeptide refers to a polypeptide comprising: are identified in one or more publicly available databases such as: HGNC:3373, Entrez Gene:2033, Ensembl:ENSG0 0000100393, OMIM:602700, UniProtKB:Q09472. In some embodiments, the EP300 polypeptide has the sequence of SEQ ID NO: 7, or SEQ ID NO: No. 7 and approximately 80% or more, approximately 85% or more, approximately 90% or more, approximately 95% or more, or approximately 98% or more or a polypeptide comprising a portion of SEQ ID NO:7. The EP300 polypeptide of SEQ ID NO: 7 represents the immature or preprocessed form of mature EP300. and therefore, the EP300 polypeptide of SEQ ID NO: 7 is referred to herein as In some embodiments, the EP300 polynucleotide comprises a mature portion or a processed portion. The peptide is a sequence of SEQ ID NO: 22 or SEQ ID NO: 23, or a sequence of SEQ ID NO: 22 or SEQ ID NO: No. 23 and approximately 80% or more, approximately 85% or more, approximately 90% or more, approximately 95% or more, or approximately 98% A polynucleotide sequence having the above homology, or SEQ ID NO: 22 or SEQ ID NO: 23 The present invention also includes polynucleotides comprising a portion of the above.

[0075] POM121C (nuclear pore membrane protein POM121) is known to be involved in the biogenesis of nuclear pores. It is believed that the membrane protein is a member of a group of proteins called pore proteins. "POM121C" as used herein refers to a member of the signaling lymphocyte activation molecule family. is the self-ligand receptor of Lee, and in humans, it refers to the polypeptide encoded by the POM121C gene. In some embodiments, the POM121C polypeptide is identified in one or more publicly available databases as follows : HGNC:34005, Entrez Gene:100101267, Ens embl:ENSG00000272391, OMIM:615754, UniProt KB:A8CG34. In some embodiments, the POM121C polypeptide is the sequence of SEQ ID NO: 8, or a polypeptide sequence having about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 98% or more homology with SEQ ID NO: 8, or a polypeptide containing a part of SEQ ID NO: 8. The POM121C polypeptide of SEQ ID NO: 8 may represent the immature or pre-processed form of mature POM 121C, and thus, the mature or processed part of the POM121C polypeptide of SEQ ID NO: 8 is included herein. In some embodiments, the POM121C polynucleotide is the sequence of SEQ ID NO: 24, or a polynucleotide sequence having about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 98% or more homology with SEQ ID NO: 24, or a polynucleotide containing a part of SEQ ID NO: 24. otide is included.

[0076] Also disclosed herein are compositions that reduce the amount of one or more transcription factors selected from the group consisting of DNAJB1 / HSP40, ATF6, ATF4, and PERK, or suppress their function. Thus, DNAJB1 / HS P40, ATF6, ATF4, and PERK polynucleotides are correlated and / or act in this specification. small activating RNAs (saRNAs), e.g., small interfering RNAs (siRNAs), and microRNA (miRNA), or CRISPR RNA, e.g., crisg Compositions containing RNA or tracr / mate RNA are included. Methods for using saRNA to reduce or inhibit the activity of a gene are known in the art. See, for example, International Publication No. 2019 / 00024, which is incorporated herein by reference in its entirety. See, for example, U.S. Pat. No. 4,863,222. Therefore, the present specification refers to the use of saRNA. Increased expression of HNF4α by using DNAJB1 / HSP40, ATF6, and ATF4 and PERK. In some embodiments, the composition comprises a D It includes nucleic acids that reduce the amount of NAJB1 / HSP40 or suppress its function. In some embodiments, the composition reduces the amount or inhibits the function of ATF6. In some embodiments, the composition reduces or inhibits the amount of ATF4. In some embodiments, the composition comprises a nucleic acid that inhibits or suppresses the function of PERK. In some embodiments, the composition comprises a nucleic acid that reduces or inhibits the function of The product further includes a nucleic acid encoding HNF4α.

[0077] DNAJB1 / HSP40 (heat shock protein 40) regulates gene expression and translation Translation initiation, folding and unfolding, and protein translocation and DNAJ / HSP is a molecular chaperone protein that may play an essential role in the degradation and repair of DNA. The activity of 40 is regulated by several post-translational modifications. 40 are phosphorylated proteins (e.g., DnaJA1, DnaJB4, DnaJC1 , DnaJC29), whose expression and function are related to acetylation (e.g., DnaJ A1, DnaJB2, DnaJB12, DnaJC5, DnaJC8, DnaJC13) , glycosylation (DnaJB11, DnaJC10, DnaJC16), palmitoylation (DnaJC5, DnaJC5B, DnaJC5G), methylation (DnaJA1-4), Prenylation (DnaJA1, DnaJA2, DnaJA4) and intramolecular disulfide The formation of bonds (DnaJB11, DnaJC3, DnaJC10) further facilitates cotranslational translation. "DNAJB1 / HSP40" as used herein refers to a gene encoding a nucleotide sequence that can be modified translationally and post-translationally. It is an autoligand receptor of the signal transduction lymphocyte activation molecule family, and in humans, It refers to the polypeptide encoded by the DNAJB1 gene. In this context, the DNAJB1 polypeptide may be identified by one or more publicly available data, such as: It is identified in the database: HGNC:5270, Entrez Gene :3337, Ensembl:ENSG00000132002, OMIM:60457 2, UniProtKB:P25685. In some embodiments, the DNAJB1 polypeptide The peptide has a sequence identical to SEQ ID NO: 9 or has a sequence identical to SEQ ID NO: 9 but is about 80% or more, about 85% or more, about 90% or more % or more, about 95% or more, or about 98% or more homology to the polypeptide sequence; includes a polypeptide comprising a portion of SEQ ID NO: 9. The DNAJB1 polypeptide of SEQ ID NO: 9 may represent an immature or preprocessed form of mature DNAJB1, and therefore, The text includes mature or processed portions of the DNAJB1 polypeptide of SEQ ID NO:9. In some embodiments, the DNAJB1 polynucleotide is SEQ ID NO:25 or SEQ ID NO: 26, or about 80% or more, about 85% or more of the sequence of SEQ ID NO: 25 or SEQ ID NO: 26 or a polynucleotide having about 90% or more, about 95% or more, or about 98% or more homology thereto. or a polynucleotide comprising a portion of SEQ ID NO:25 or SEQ ID NO:26. nothing.

[0078] ATF6 is a protein-binding protein that mediates the unfolded protein response (UNSP). They are sensors of the UPR (Unified Photon Response) and function to regulate transcriptional expression. In this case, ATF6 is transported from the endoplasmic reticulum (ER) to the Golgi apparatus upon ER stress. where it is proteolytically cleaved to regulate protein folding and transport. It was found that it releases the N-terminal ATF6 segment, which is a transcription factor for genes involved in "ATF6" as used herein refers to a member of the signaling lymphocyte activation molecule family. It is an autoligand receptor for β-amyloid, and in humans is encoded by the ATF6 gene In some embodiments, the ATF6 polypeptide is one of the following: Identified in one or more publicly available databases: HGNC :791, Entrez Gene:22926, Ensembl:ENSG00000 118217, OMIM:605537, UniProtKB:P18850. Several In this embodiment, the ATF6 polypeptide has the sequence of SEQ ID NO: 10, or a sequence identical to SEQ ID NO: 10. Approximately 80% or more, approximately 85% or more, approximately 90% or more, approximately 95% or more, or approximately 98% or more The polypeptide sequence may be a polypeptide having the same sequence as, or containing a portion of, SEQ ID NO:10. The ATF6 polypeptide of SEQ ID NO: 10 represents the immature or preprocessed form of mature ATF6. The mature portion of the ATF6 polypeptide of SEQ ID NO: 10 may also be included herein. In some embodiments, the ATF6 polynucleotide comprises: The sequence of SEQ ID NO: 27, or a sequence identical to SEQ ID NO: 27 by about 80% or more, about 85% or more, or about 90% or more , a polynucleotide sequence having about 95% or more, or about 98% or more homology thereto; It includes a polynucleotide comprising a portion of SEQ ID NO:27.

[0079] ATF4 enhances the transcriptional expression of genes involved in amino acid metabolism and oxidative stress resistance. It is a transcriptional activator of UPR target genes that may play a role in 6) “ATF4” is used herein to refer to an autoantibody of the signaling lymphocyte activation molecule family. It is an autoligand receptor and in humans is encoded by the ATF4 gene. In some embodiments, the ATF4 polypeptide is one or more of the following: or identified in multiple publicly available databases:HGNC:7 86, Entrez Gene:468, Ensembl:ENSG000001282 72, OMIM:604064, UniProtKB:P18848. Some implementations In some embodiments, the ATF4 polypeptide has the sequence of SEQ ID NO:11, or a sequence that is about 80% identical to SEQ ID NO:11. or more, about 85% or more, about 90% or more, about 95% or more, or about 98% or more homology or a polypeptide comprising a portion of SEQ ID NO: 11. The 11 ATF4 polypeptides may represent immature or preprocessed forms of mature ATF4. Thus, the mature portion of the ATF4 polypeptide of SEQ ID NO: 11 or In some embodiments, the ATF4 polynucleotide comprises a processing portion. 28, or about 80% or more, about 85% or more, about 90% or more, about 95% or more of the sequence of SEQ ID NO: 28 % or more, or about 98% or more homology to the polynucleotide sequence of SEQ ID NO: The polynucleotide comprises a portion of 28.

[0080] PERK (protein kinase RNA-like endoplasmic reticulum kinase) is typically It is activated by the recruitment of chaperones from the cytosolic kinase domain, resulting in oligomerization of the kinase domain. It is a type 1 transmembrane protein that mediates the activation and function of PERK and ATF4. CCAAT enhancer is a key protein mediating proapoptotic signaling in the liver Cholesterol-binding protein (C / EBP) homologous protein (CHOP), which is a major cause of liver disease It is involved in the progression of peritoneal carcinoma (Malhi H et al., 2011). PERK is also involved in the progression of peritoneal carcinoma (PDC) by inhibiting the EIF2AK3 , also known as ", and are referred to herein as members of the signaling lymphocyte activation molecule family. It is an autoligand receptor and in humans is encoded by the EIF2AK3 gene. In some embodiments, the PERK polypeptide refers to a polypeptide that: identified in one or more publicly available databases: NC :3255, Entrez Gene:9451, Ensembl:ENSG00000 172071, OMIM:604032, UniProtKB:Q9NZJ5. Several In this embodiment, the PERK polypeptide has the sequence of SEQ ID NO: 12, or a sequence identical to SEQ ID NO: 12. Approximately 80% or more, approximately 85% or more, approximately 90% or more, approximately 95% or more, or approximately 98% or more The polypeptide sequence may be a polypeptide having the same sequence as, or containing a portion of, SEQ ID NO:12. The PERK polypeptide of SEQ ID NO: 12 represents the immature or preprocessed form of mature PERK. and the mature or processed portion of the PERK polypeptide of SEQ ID NO: 12 may be used herein. In some embodiments, the PERK or EIF2AK3 polynucleoside moiety is The peptide may be a sequence of SEQ ID NO: 29 or SEQ ID NO: 30, or a sequence of SEQ ID NO: 29 or SEQ ID NO: No. 30 and approximately 80% or more, approximately 85% or more, approximately 90% or more, approximately 95% or more, or approximately 98% A polynucleotide sequence having the above homology, or SEQ ID NO: 29 or SEQ ID NO: 30 The present invention also includes polynucleotides comprising a portion of the above.

[0081] The composition of any preceding embodiment may further comprise an HNF4α agonist, NF4α agonists are disclosed in U.S. Pat. No. 6,233,629, which is incorporated herein by reference for all purposes. As more fully described in patent application publication no. 2014 / 0249209. It is meant to refer to compositions thereof.

[0082] In some embodiments, the composition and / or vector of any preceding aspect is In some embodiments, the bioavailable formulation may be prepared using a physicochemically acceptable carrier. A physiologically acceptable carrier may be used to transport the composition and / or vector within a host cell and / or In some embodiments, a biologically acceptable carrier can be introduced into the host cell. The body may deliver the composition and / or vector to target cells, such as hepatocytes in the liver of the subject, and / or or between target cells. Importantly, in some embodiments, The compositions and / or vectors may be combined with a biologically acceptable carrier, such as a functional polymer, For example, DNA and RNA can be transported into the nucleus of target cells (eg, hepatocytes).

[0083] Treatment methods Herein, the expression of the transcription factor HNF4α in the nucleus of hepatocytes in a subject and / or or methods of treating liver disease by increasing transport and / or retention are provided. In some embodiments, the method comprises detecting PROX1, NR5A2, NR0B2 , MTF1, SREBP1, EP300, and POM121C. upregulating the expression or function of one or more transcription factors associated with the / or a group consisting of DNAJB1 / HSP40, ATF6, ATF4, and PERK downregulating the expression or function of one or more transcription factors selected from In some aspects, the present invention provides a method for treating liver disease in a subject in need thereof. A method of treating, comprising administering to a subject a vector, wherein the vector is , NR5A2, NR0B2, MTF1, SREBP1, EP300, and / or PO M121C, and one or more transcription factors selected from the group consisting of functional fragments thereof. In some embodiments, methods are disclosed herein that include nucleic acids encoding the 1. A method of treating liver disease in a subject in need thereof, comprising administering to the subject a DNAJB1 / H one or more selected from the group consisting of SP40, ATF6, ATF4 and PERK a method comprising administering a composition that downregulates the expression or function of a transcription factor of In some embodiments, the composition comprises siRNA, miRNA, sg In another embodiment, the method comprises the step of: Increasing acetylation of HNF4α at ys106 increases cMET expression and / or increase AKT activation via phosphorylation at Thr308. Includes:

[0084] In some embodiments, provided herein are methods for treating liver disease in a subject in need thereof. The method includes administering a vector to a subject, wherein the vector encodes PROX1. In some embodiments, methods are disclosed herein that include loading nucleic acids. A method of treating liver disease in a subject in need thereof, comprising administering to the subject a vector. and wherein the vector comprises a nucleic acid encoding SREBP1. In some embodiments, the vector further comprises a nucleic acid encoding HNF4α. In some embodiments, the vector encodes HNF4α, PROX1, and SREBP1. In some embodiments, the method comprises one or more nucleic acids encoding HNF. The method further comprises administering a vector comprising a nucleic acid encoding 4α.

[0085] As mentioned above, PROX1, NR5A2, NR0B2, MTF1, SREBP1, EP 300, and POM121C all have functions that, for example, regulate HNF4α acetylation, cellular metabolic pathways, and Through direct or indirect mechanisms, including the formation of tracts or nuclear pore complexes, HNF4 These factors are transcription factors and / or regulatory factors that control nuclear transport of HNF4α. Such effects can restore hepatocellular function in patients with liver disease.

[0086] Thus, in some embodiments, administration of one or more vectors results in a In some embodiments, the vector (or vectors) increases the amount of HNF4α in the nuclei of hepatocytes. Administration of one or more of the following compounds does not increase the total amount of HNF4α in hepatocytes. Administration of the vector(s) increases the total amount of HNF4α in hepatocytes. In some embodiments, the vector of any preceding embodiment further comprises a nucleic acid encoding HNF4α. In some embodiments, the vectors disclosed herein contain an HNF4α isoflavon. In some embodiments, the nucleic acid encoding the HNF form 2 polypeptide is further included. The 4α isoform 2 polypeptide has at least about 80%, about 85%, or about the same sequence as SEQ ID NO:1. 90%, about 95%, or about 98% identical sequences or fragments thereof. In some embodiments, the nucleic acid has at least about 80%, about 85%, about 90%, about 95% identity with SEQ ID NO: 31. , or about 98% identical, or a fragment thereof.

[0087] Thus, in some aspects, provided herein are methods for treating liver disease in a subject in need thereof. A method for treating a disease comprising the steps of: The present invention also provides a method for administering a compound to a subject in need thereof. 1. Use of a composition for preparing a medicament for treating liver disease in a subject, comprising: administering a composition to a subject, wherein the composition encodes HNF4α isoform 2. The present invention also includes uses of nucleic acids.

[0088] The vectors used in this method include plasmids, bacteriophages, and virus particles ( isolated, attenuated, recombinant, etc.), exosomes, extracellular vesicles, and In some embodiments, the nanoparticles may be any of those described herein, including nanoparticles. In some embodiments, the vector is a plasmid. In some embodiments, the vector is a viral particle. In some embodiments, the vectors are naturally occurring and / or engineered capsids. In some embodiments, the vector is a viral vector having an exosome. In some embodiments, the vector is a nanoparticle. In some embodiments, the vector is an mRNA lipid nanoparticle. (e.g., closed-ended DNA (ceDNA) or RNA. ceD Methods for making and using NA and ceDNA are known in the art. See, for example, International Publication No. WO 2014 / 013994, which is incorporated herein by reference in its entirety. Pamphlet No. 9 / 169233 and Pamphlet No. WO 2017 / 152149 For methods, materials, delivery nanoparticles, and amounts and formulations, see the accompanying documents. nanoparticles, components thereof, and delivery of such components, including their manufacture and use With respect to general information, all of which is useful in the practice of the invention and may be consulted for any purpose. Wu et al., J. Biol. Chem. 262, 4429, 1987, USA, incorporated herein by reference. Patent Application Publication No. 2011 / 0274706 and International Publication No. 2018 / 1704 It is mentioned in the brochure issue 05.

[0089] In some aspects, provided herein are methods for treating liver disease in a subject in need thereof. The method includes administering a composition to a subject, the composition comprising: 0, ATF6, ATF4, and PERK. Methods are disclosed for reducing the amount of or inhibiting the function of a transcription factor.

[0090] As mentioned above, DNAJB1 / HSP40, ATF6, ATF4, and PERK All are transcriptional regulators of endoplasmic reticulum (ER) stress. The ER is responsible for the proper follicular organization of proteins. A type of membranous organelle in eukaryotic cells that is important for binding, modification, and transport ER stress occurs when the ER's capacity to fold proteins is saturated. These transcription factors can induce responses such as cell death and inflammation. These translocations are regulated by a pathway related to HNF4α. Reducing the amount or inhibiting the function of one or more transcription factors can be used to treat liver cancer. It has been shown that it can restore liver cell function in patients with the disease. In some embodiments, administration of the composition increases the amount of HNF4α in the nuclei of hepatocytes in the subject. In some embodiments, administration of the composition increases the total amount of HNF4α in hepatocytes. In some embodiments, administration of the composition does not increase the total amount of HNF4α in hepatocytes. In some embodiments, the composition further comprises a nucleic acid encoding HNF4α. include.

[0091] In some embodiments, the compositions inhibit DNA damage by knocking down these genes. Selected from the group consisting of JB1 / HSP40, ATF6, ATF4, and / or PERK It reduces the amount of one or more transcription factors that are involved in the transcription of ATP. 6, ATF4, and / or PERK knockdown inhibited DNAJB1 / HSP40, mRNA encoding ATF6, ATF4, and / or PERK, or DNAJ B1 / HSP40, ATF6, ATF4, and / or enzymes required for PERK activity RNA interference occurs when a gene is recognized by a complementary RNA molecule of related mRNA, such as the mRNA that encodes it. For example, the transcription factor may be mediated by interfering RNA (RNAi). The molecule to be delivered is delivered to the host via a suitable vector, such as via transfection or transduction. - e.g., hepatocytes by lentiviral or retroviral vectors or nanoparticles or may be introduced into hepatocyte precursors.

[0092] In some embodiments, DNAJB1 / HSP40, ATF6, ATF4, and / or or PERK. Any one or more CRISPR complex components for the purposes disclosed herein Administered with or within a viral particle, virion, or viral vector In some embodiments, the sgRNA or tracr / mate R NAs can be packaged with one or more reprogramming factors In some embodiments, the viral particle, virion, or viral vector The encapsulated sgRNA molecule is packaged with one or more reprogramming factors. Methods, materials, delivery vehicles, vectors, particles, AAV, and amounts and CRISPRCas systems, including their manufacture and use in relation to formulations, etc.; All general information regarding the ingredients and delivery of such ingredients is contained in this patent. U.S. Pat. No. 6,629,493, which is incorporated herein by reference for all purposes and is useful in the practice of the invention. Reference is made to patent application publication no. 2018 / 0057839.

[0093] As mentioned above, "liver disease" as used herein generally refers to any condition that affects the liver. refers to diseases, disorders, and conditions that cause liver damage, such as simple accumulation of fat in liver cells (steatosis), macrocytosis, Steatosis, periportal and lobular inflammation (steatohepatitis), cirrhosis, fibrosis, hepatic ischemia, hepatocytes A wide range of conditions including liver cancer, early stage liver disease, end stage liver disease, and liver failure The severity of the disease can vary. Thus, steatosis, macrovesicular steatosis, steatohepatitis, cirrhosis, fibrosis , liver cancer, hepatocellular carcinoma, end-stage liver disease, chronic liver disease, and liver failure are all classified under the category of "liver disease." The degree or severity of "cirrhosis" is determined by the Child-Pugh score. The classification includes five clinical items: total bilirubin, serum albumin, The levels of hepatic encephalopathy, prolonged prothrombin time, ascites, and hepatic encephalopathy were measured at various levels for each clinical item. Each item is scored using a scoring system with values of 1, 2, and 3 corresponding to the level. The most stringent level is assigned a value of 3 points. The total points for all five items are added together. A Child-Pugh score and classification are reached by scoring 5-6. A score of 7 to 9 is designated as Child-Pugh Class B, and a score of 1 is designated as Child-Pugh Class B. A score of 0 to 15 is designated as Child-Pugh Class C. Class A indicates the least severe liver disease, and Child-Pugh class C indicates the most severe. In some embodiments, the methods disclosed herein are indicative of severe liver disease. Have Child-Pugh class B liver disease or Child-Pugh class BC liver disease In some embodiments, the methods disclosed herein can be used to treat a subject. A method for treating a subject with Child-Pugh class A liver disease In some embodiments, the liver disease is alcoholic hepatitis. In some embodiments, the methods disclosed herein involve ex vivo transplantation of an ischemic donor liver. The present invention can be used to treat liver damage by ivo perfusion before or after liver resection. It can be used to treat liver cancer before or after cancer treatment, including The early stage of liver disease is non-alcoholic fatty liver disease (NAFLD), Nonalcoholic steatohepatitis (NASH), including but not limited to fatty liver, alcoholic hepatitis, and alcohol It is understood that the condition may be alcohol-related liver disease, including alcohol-related cirrhosis, and End-stage liver disease as disclosed herein includes, for example, The present invention relates to the treatment of idiopathic rheumatoid arthritis, including viral, alcoholic, non-alcoholic, and cryptogenic rheumatoid arthritis. It should also be understood that this may be due to any cause known in the art.

[0094] Thus, the methods disclosed herein are directed to a patient having liver disease of any of the preceding aspects. It can be used to improve liver function in a subject. Such improvement in liver function can be achieved, for example, by , increased serum albumin, decreased serum ammonia levels, decreased total bilirubin, and encephalopathy scores. This can be indicated by an increase in blood glucose level and / or a decrease in prothrombin time. Therefore, the methods disclosed herein increase serum albumin levels and It reduces ammonia levels, reduces total bilirubin levels, increases encephalopathy scores, and and / or prothrombin time prolongation.

[0095] Liver disease has multiple stages, including inflammation, fibrosis, cirrhosis, end-stage liver disease, and liver cancer. It can progress in chronic infection with hepatitis viruses, alcohol-mediated cirrhosis, and / or Chronic liver disease, including nonalcoholic steatohepatitis (NASH), is known to have multiple causes. Because the timing of liver disease is often unpredictable, The disclosed methods of preventing, reducing, and / or inhibiting inflammation, fibrosis, cirrhosis, end-stage liver disease, It can be used before or after the onset of liver disease and / or liver cancer, and also for the treatment of hepatitis virus infections. , alcohol-mediated cirrhosis, and / or nonalcoholic steatohepatitis before or during It can be used to treat, prevent, inhibit, and / or slow any stage of liver disease. It is understood that the disclosed methods can be used to treat inflammation, fibrosis, cirrhosis, It can be performed any time before the onset of end-stage liver disease and / or liver cancer. In embodiments, the disclosed methods include treating inflammation, fibrosis, cirrhosis, end-stage liver disease, and / or liver damage. 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49 , 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 2 2, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 year ago; 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 month ago; 30, 29, 28, 27, 26, 25, 24, 23, 22 , 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8 , 7, 6, 5, 4, or 3 days ago; 60, 48, 36, 30, 24, 18, 15, 12, Can be used 10, 9, 8, 7, 6, 5, 4, 3, or 2 hours before use; or contributes to the development of inflammation, fibrosis, cirrhosis, end-stage liver disease, and / or liver cancer. , 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 , 60, 75, 90, 105, 120 minutes later; 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 24, 30, 36, 48, 60 hours later; 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 45, 60, 90 days or more After 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more; 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 4 6, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33 , 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, It can be used after 4, 3, 2, or 1 year.

[0096] Liver resection is recommended for patients with liver disease (e.g., cirrhosis, end-stage liver disease, and / or liver cancer). The disclosed method involves the surgical removal of all or part of an elephant's liver before or after hepatectomy. In one aspect, the disclosed method can be performed on a subject at any time after a liver resection procedure. 5, 4, 3, 2, or 1 year before surgery; 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 month ago; 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 2 0, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 days ago; 60, 48, 36, 30, 24, 18, 15, 12, 10, 9, 8 It can be used 7, 6, 5, 4, 3, or 2 hours before liver resection; or , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 , 50, 55, 60, 75, 90, 105, 120 minutes later; 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 24, 30, 36, 48, 60 hours later; 3, 4, 5, 6 ,7,8,9,10,11,12,13,14,15,16,17,18,19,20, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 45th, 60th, 90th, or more; after 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more After; 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48 , 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 2 1, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7 It can be used after 6, 5, 4, 3, 2, or 1 year.

[0097] The vectors or compositions described herein can be administered orally, topically, intravenously, subcutaneously, transdermally, or intravenously. utaneous, transdermal, intramuscular, intraarticular, parenteral, intraarterial, intradermal, ventricular by any route, including intravenous, intracranial, intraperitoneal, intralesional, intranasal, intrarectal, and intravaginal, The compound can be administered to a subject parenterally or via an implanted reservoir. The term includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, and intralesional. In some embodiments, the vector is administered intravenously or intraperitoneally, including intravenously, intravenously, intracranially, and intracranially. Alternatively, administration of the composition is intravenous.

[0098] Another aspect of the present disclosure is a method for detecting and detecting the presence of PROX1, NR5A2, NR0B2, MTF1, SREBP1 , EP300, and / or POM121C, and functional fragments thereof. The composition described above that increases the amount or function of one or more selected transcription factors, and DNA AJB1 / HSP40, ATF6, ATF4, and PERK Both compositions that decrease the amount of or inhibit the function of one or more transcription factors In some embodiments, both compositions are administered simultaneously. In other embodiments, one composition is administered before the other. In any preceding embodiment, the method further comprises upregulating HNF4α expression or function. In some embodiments, the method of any preceding aspect further comprises: and the further administration of a 4α agonist, and this term shall be used for all purposes. No. 2014 / 0249209, incorporated herein by reference. The present invention refers to the compositions described more fully in

[0099] The frequency of administration of the vector or composition of any preceding embodiment includes, but is not limited to, , at least once a year, once every two years, once every three years, once every four years, once every five years, 6 Once per year, once per seven years, once per eight years, once per nine years, once per ten years, at least Once every 2 months, once every 3 months, once every 4 months, once every 5 months, once every 6 months, Once every 7 months, once every 8 months, once every 9 months, once every 10 months, once every 11 months times, at least once a month, once every three weeks, once every two weeks, once a week, twice a week, three times a week, Administration can also be continuous, including four times per week, five times per week, six times per week, or daily. The compound levels can be adjusted to maintain them within any desired specified range. To treat liver disease using the vector and / or composition of any preceding embodiment As used herein, the terms "administration" or "administering" include, for any purpose, No. 2018 / 0057839, which is incorporated herein by reference. Included are those forms of administration that are more fully described herein. [Example]

[0100] The following examples are provided to illustrate compositions, methods, and results in accordance with the disclosed subject matter. These examples include all aspects of the subject matter disclosed herein. These are not intended to be exhaustive but to illustrate representative methods and results. The examples are not intended to exclude equivalents and variations of the invention that are obvious to one skilled in the art. do not have.

[0101] [Example 1] Methods and Materials. Human Samples and Hepatocyte Isolation. De-identified normal human liver tissue and / or cells are isolated. After obtaining written informed consent, the liver tissue cells were Distribution System (Pittsburgh, PA) University of Pittsburgh funded by NIH Contract No. HSN276201200017C Approved by the Human Research Review Committee of the University of Tokyo Adult human liver tissue and / or cells were also obtained by the Pittsburgh Department of Hepatology. The University's Human Research Review Committee and Ins National Review Board (IRB) The study was conducted under a protocol approved by the UPMC Research Institute (IDI) under the National Institutes of Health (NIH) Number PRO12090466. Hepatocytes were obtained from the Ira J Fox Laboratory at the Children's Hospital of New York (Table 1). The cells were isolated using a three-step collagenase digestion technique as previously described (Gramignoli R After isolation, cells were purified using trypan blue exclusion as previously described. Cell viability was assessed and only preparations with cells >80% viability were used for analysis.

[0102] [Table 1]

[0103] In silico HNF4α post-translational modification (PTM) analysis. Modulation of HNF4α cellular localization. A computer search of databases and publications was performed to identify PTMs that support the The in silico analysis was carried out via the ELISA (Figure 5). The process was divided into three phases: identification, screening, and The study was divided into screening and selection. First, 51 PTMs were identified. During the screening phase, 23 PTMs were selected by applying two exclusion criteria (Figure 5). The two most reliable PTMs for HNF4α localization that can be evaluated are We identified one phosphorylation and one acetylation modification.

[0104] Stable isotope analysis using gas chromatography-mass spectrometry. One million human liver cells The cells were incubated with 13-C6-labeled glucose and glucose in Dulbecco's modified Eagle's medium F12. The cells were cultured for 96 hours in the presence of glutamine isotope tracer (Thermo Fisher Scientific). The medium was removed and the cells were placed on ice-cold lyophilized ice. The cells were then washed with phosphate-buffered saline. The cells were then placed in a 400 μL solution containing 1 μL of norvaline. Quench with 400 μL of methanol and 400 μL of water, scrape, and transfer to 800 μL of ice-cold Wash with chloroform and vortex for 30 min at 4 °C, 7,300 rpm, 4 °C for 10 min. The upper aqueous phase was collected for metabolite analysis. Metabolite extracts were analyzed by centrifugation at 14,0 Centrifuge at 00g for 10 minutes to separate the polar phase, protein interphase, and chloroform phase. The water / methanol phase containing the polar metabolites was transferred to a new microcentrifuge tube and Dry in dVac and store for 8 min until gas chromatography-mass spectrometry (GC-MS) analysis. The mixture was stored at 0° C. Then, 30 μL of methoxyamine hydrochloride (Thermo Scientific The dried sample was added to the dried sample and incubated at 30°C for 2 min with intermittent vortex mixing. A total of 45 μL of MBTSTFA + 1% tert-butyldimethyl ether was added. Trichlorosilane was added to the sample and incubated at 55°C for 1 hour. The sample was transferred to a gas chromatography (GC) vial using a glass insert and The GC-MS analysis was performed using an Agilent 5977B Agil equipped with a 30 m HP-5MSUI capillary column connected to a mass spectrometer Polar metabolism was performed using a ent 7890GC (Santa Clara, CA). For the samples, the following heating cycle for the GC oven was used: 100 °C for 3 min, then This was followed by a constant ramp of 5°C / min to 300°C, with a total run time of 48 min at 300°C. Data were acquired in scan mode. Relative abundance of metabolites was determined for each metabolite fragment. Calculated from the integrated signal of all potentially labeled ions per piece. Before the analysis in the laboratory, the mass distribution of the isotopically substituted compounds was analyzed using IsoCorrecto®. Metabolite levels were normalized to the signal of the internal standard, norvaline. Normalized fractional enrichment calculations represent the fractional contribution of 13C from substrate to intermediate metabolite. This is calculated as follows:

[0105]

number

[0106] Immunohistochemistry and quantification of HNF4α. Paraffin-embedded liver tissue was dextran-dextran-dextran-dextran. The water was removed using a solvent and then dehydrated with ethanol. Antigen unmasking was performed by rinsing the slides in 3% hydrogen peroxide. Incubate, block with normal animal serum, and then incubate with primary antibody overnight at 4°C. The primary antibodies used are listed in Table 2. The tissue sections were then incubated with Biotinylated secondary antibodies (BA-1000; Vector Lab) corresponding to the primary antibody species were used. Incubate with PBS (Burlingame, CA) for 3,3 '-Diaminobenzidine (SK-4105; Vector Laboratories) Peroxidase activity was visualized by exposure to HCl. Counterstaining was performed using the Richard-Allen method. The Scientific Signature Series Hematoxylin Quantification was performed using a fluoroscopy (Microfluidics Laboratory, San Diego, CA, USA). Nuclear and cytoplasmic HNF4α immunoreactivity was assessed independently by two liver pathologists. , 1,000 hepatocytes counted per sample in three high-power fields were used for grading. These analyses included normal liver (n = 2), Child-Pugh B (n = 4), and Child-Pugh C (n=2) was included. Child-Pugh B and C were included. The results are presented as a percentage of the total number of cells counted. It is represented as a page.

[0107] [Table 2]

[0108] Protein extraction and Western blotting to perform protein expression analysis The isolated hepatocytes were then divided into two fractions, one of which was cultured using a standard method as previously described. The other fraction was used for nuclear protein extraction according to the procedure (Bell AW et al., 2006). For nuclear protein isolation, 1 × 10 cells per patient were used. 7 From 5 x 10 7 Wash the isolated hepatocytes between 100 ml and 100 ml of 40 mmol / L Tris (pH 7.6), 100 ml of ... The samples were collected in 4 mmol / L NaCl and 1 mmol / L EDTA, then centrifuged. The cell pellet was then centrifuged (5 min, 100 g). The cell pellet was then centrifuged in 2 mL of hypotonic buffer [protease and and phosphatase inhibitor cocktail (Sigma, St. Louis, MO). mmol / L HEPES (pH 7.9), 10 mmol / L NaH2PO4, 1.5 mmol / L MgCl2, 1 mmol / L DTT, 0.5 mmol / L spermidine The cells were suspended in 1 mol / L NaF and 1 mol / L NaF. After incubation on ice for 10 minutes, The samples were homogenized in an Ounc homogenizer and then centrifuged (5 min, 800 g). Cell lysates were monitored using trypan blue staining. The supernatant was collected as an intracytoplasmic extract. The nuclear pellet was washed two additional times with the same buffer.

[0109] Nuclear proteins were incubated in 50-100 μL of hypertonic buffer [protease and phosphatase inhibitors]. 30 mmol / L containing enzyme inhibitor cocktail (Sigma, St. Louis, MO). HEPES (pH 7.9), 25% glycerol, 450 mmol / L NaCl, 12 mmol / L MgCl2, 1 mmol / L DTT, and 0.1 mmol / L ED The extract was extracted with 1000 ml of ethanol at 30,000 g for 45 minutes at 4°C with continuous stirring. After centrifugation, the supernatant was collected and resuspended in the same solution except that it contained 150 mmol / L NaCl. The protein concentration was determined by bicinchoninic acid assay (Sigma, St. Louis, MO).

[0110] Western blot analysis was performed according to standard procedures ( Natarajan A et al., 2007 The intensity of each protein band was calculated using the National Institutes of Health Quantification was performed using Image J software from the University of California. The primary antibodies and their dilutions are listed.

[0111] RNA sequencing and analysis. Whole genome strand-specific RNA sequencing (RNA-seq) was used to RNA expression levels were profiled from isolated human primary hepatocytes. The sequence library was prepared as previously described (Hainer SJ et al., Genes Dev. 2015) and RNA was prepared as described in the literature (Kumar R et al., 2012). l from intestinal cells and subsequently purified on a column according to the manufacturer's instructions (Zym o RNA Clean and Concentrator Column). From total RNA, rRNA is extracted using this Pooled antisense oligo hybridization and and RNase H digestion-mediated depletion (Morlan JD et al., 2012; Adic onis X et al., 2013). On Zymo RNA Clean and Concentrator columns. After purification, the first strand cDNA was synthesized. Then, the second strand cDNA was synthesized. Purified and fragmented RNA-seq libraries were prepared using Illumina technology. Briefly, end repair, A-tailing, and barcoded adapter insertion were performed. Ligation followed by PCR amplification and size selection. The quality of the fragments was assessed by quBit quantification and fragment size distribution evaluation of approximately 10 fragments from each library. The libraries were analyzed by paired-end PCR and confirmed by Sanger sequencing. Sequenced using lumina sequencing.

[0112] Paired-end reads were analyzed using QIAGEN's CLC Genomics workbench Align to hg38 using TPM (transcript per million) To sort the data, we used K-means clustering. was run using Cluster 3.0 (De Hoon MJ et al., 2004) and Java Heatmaps were created using TreeView (Saldanha AJ, 2004). Chi (Murphy SL et al., 2015), insertion cost (Goldman L et al., 2016), and deletion cost (Goldman L et al., 201 6) The default settings were used. Ingenuity Pathway Analysis (IPA) was used. to identify differentially expressed genes, predict downstream effects, and identify targets (QIAGEN Bioinformatics;www.qiagen.com / ingenuity) Using regulatory action analysis within IPA, we identified the relationship between upstream regulators and biological functions. The default settings were used in the analysis (i.e., upstream regulators were identified as genes RNA-seq data were collected from Gene Explorer. Available at the NBS Omnibus (accession no. www.ncbi.nlm.nih.gov / nbs / nbs / accession_no_2016010 ...). ih.gov / geo / query / acc.cgi?acc=GSE134422).

[0113] AKT inhibition in normal human hepatocytes. Normal human hepatocytes (1 million cells / well) were cultured in a 2000-well plate. Cells were cultured on collagen-coated wells. The cells were then cultured for 6 hours in the presence of 5 μM MK-2206 ( Cayman Chemical, Ann Arbor, Michigan) 24 hours a day Total, cytoplasmic and nuclear proteins were analyzed using the wells as described above. Extracted for stan blotting.

[0114] Statistical analysis. Data were expressed as mean ± SD. Western blot of two statistical groups The results from the study were evaluated by the Mann-Whitney nonparametric test, and three statistical The results of all groups were evaluated by the Kruskal-Wallis nonparametric test. Comparisons of the proteins were performed by Dunn's multiple comparison test. Aman's rank correlation test was used to evaluate the correlation between protein expression and chi- Relationship with clinical status measured as Child-Pugh score and MELD score Statistics were performed using Prism 4.0 (GraphPad Software Informatics). c., San Diego, California, USA). P< Differences were considered significant when the value was 0.05.

[0115] To identify direct dependencies between proteins analyzed by Western blot, Path analysis (structural equation modeling) was used. Path analysis models are used to compare the dependent variable with multiple independent variables. The aim is to explain possible causal relationships between observed correlations between the The path model was tested and the research framework and regression weights and model flows were analyzed. Based on the fitting results, the data was modified by adding and removing paths. is plotted as a line diagram showing the direct and indirect effects of variables on the study system. The degree of correlation and linear relationship between variables is shown with P<0.05 and a level of significance. The path solution is determined by an arbitrary coefficient (the larger the number, the greater the relationship). The analysis was performed using InfoStat version 2013 (Grupo InfoStat, FCA, Universidad Nacional de Cordoba, Cordoba, This was carried out using the Argentina.

[0116] Unsupervised multivariate principal component analysis (PCA) was applied to the Western blot data to identify the We elucidated a group of proteins that distinguished between clinical conditions. Principal components (PCs) that explained most of the variance ) scatter plots were drawn. For PCA analysis, statistical software JMP version 14 ( SAS Institute, Cary, NC, USA) was used.

[0117] [Example 2] Nuclear localization of HNF4α is decreased, whereas cytoplasmic localization is increased, in human livers with end-stage liver failure. Add HNF4α functions as a transcription factor, and its nuclear localization is required for its activity (Babeu JP et al., 2014;Chellappa K et al., 2012;Guo H, 2014;Hong YH et al., 2003;Lu H et al., 2016, Song Y et al., 2015;Soutoglou E et al., 2000;Sun K et al., 2007;Yokoya ma A et al., 2011;Zhou W et al., 2012;Bell AW et al., 2006;Kritis AA et al., 1996;Tanaka T et al., 2006;Walesky C et al., 2015). Therefore, the affected liver Immunohistochemistry and Western blots were performed on hepatocytes from the specimens to detect HNF4α. The localization of IL-17 was determined and its expression correlated with hepatic decompensation. Approximately 78% of hepatocytes in the rats express HNF4α only in the cytoplasm or in the cytoplasm and weakly In contrast to normal human liver, 75% of hepatocytes expressed strong HNF4α. Total HNF4α protein expression from isolated hepatocytes was measured by Western blot analysis. As assessed by lot, no statistical differences were found between end-stage livers and normal controls. (P = 0.166; Figure 1A). This result was not surprising. The authors compared liver tissue samples from patients with degenerative diseases and controls based on the degree of functional decompensation. The ability to discern differences in HNF4α expression in patients will require the study of larger cohorts of patients. (Guzman-Lepe J et al., 2018). However, in this study, Statistically significant differences were observed based on HNF4α. HNF4α was significantly elevated from normal controls. High levels of ATP were detected in the cytoplasm of hepatocytes isolated from functionally decompensated livers compared with isolated hepatocytes from non-compensated livers. was detected in the nucleus (P = 0.023; Fig. 1B) and at lower levels in the nucleus (P = 0.0 23; Figure 1C).

[0118] Because the function and stability of HNF4α are regulated by numerous post-translational modifiers (Chellappa K et al., 2012;Guo H, 2014;Hong YH et al., 2003;Lu H et al., 2016 , Song Y et al., 2015;Soutoglou E et al., 2000;Sun K et al., 2007;Yokoyama A et al., 2011; Zhou W et al., 2012), and furthermore, its nuclear localization is important for its activity. Therefore, we performed in silico analysis to determine which modifiers regulate HNF4α localization. We found that activation of AMPKα regulates HNF4α transcription. (Hong YH et al., 2003). In addition, acetylation of HNF4α is mediated by the AKT pathway. This stabilizes the molecule and favors its retention in the nucleus (Soutoglou E et al., 2000) (Figure 5).

[0119] [Example 3] HNF4α is a key regulator of human hepatocyte function in progressive liver disease. Next, human hepatocytes from normal controls (n=4) and those with cirrhosis and end-stage liver failure were cultured. Comparison of gene expression differences between those recovered from patients (n=4) (Child-Pugh C) To evaluate and compare the study of NASH and alcohol-mediated Laennec cirrhosis Hierarchical clustering of RNA-seq data revealed that patients with liver cirrhosis and Three major dynamic patterns associated with liver dysfunction and K-means clustering The calculated values (log2FC relative to control; K=3) were elucidated. Control human hepatocytes Moderately to highly upregulated in hepatocytes from patients with end-stage liver failure compared with Representative examples of the genes evaluated are clusters I (3478 genes) and III. Most of the genes in these clusters were identified as It was associated with phagocytosis and apoptosis signaling (data not shown).

[0120] However, cluster II was significantly downregulated in end-stage hepatocytes. It consists of 1669 genes, including serine-threonine protein kinase (AKT1), Cytochrome P450 (cytochrome P450 [CYP] c8, CYP2c9, CYP2e1 , CYP3A4), and hepatocyte nuclear factor 4α (HNF4α, forkhead box a1 [ The cluster contained genes encoding the α- and β-actin-dependent pathways (e.g., FOXa1, FOXa2, FOXa3, FOXa4, FOXa5, FOXa6, FOXa7, FOXa8, FOXa9, FOXa10, FOXa11, FOXa12, FOXa13, FOXa14, FOXa15, FOXa16, FOXa17, FOXa18, FOXa farnesoid X receptor / retinoid X receptor (RXR) and liver X receptor / RX including RXR activation, mitochondrial dysfunction, oxidative phosphorylation, and inhibition of RXR function. Pathway analysis of these downregulated genes revealed that HNF4α The heatmap shows that α-glucanase was a key upstream regulator of NASH and alcohol consumption. Gene expression profiles of hepatocytes from patients with leukocyte-mediated Laennec cirrhosis These results confirm the many similarities between the two groups (data not shown). The gene expression profile of rat hepatocytes recovered from cirrhotic livers was nearly identical to that of rat hepatocytes recovered from cirrhotic livers. (Liu L et al., 2012).

[0121] [Example 4] cMET and AKT phosphorylation was significantly increased in human hepatocytes from patients with end-stage liver failure. Correlates with the nuclear localization of F4α EGFR and cMET have been identified in silico and as important modulators of HNF4α. and RNA-seq analysis, which showed that it regulates the AMPK and AKT pathways. (Komposch K et al., 2015;Paranjpe S et al., 2016;Tsagianni A et al, 2 018), performed antibody-based assays for these molecules in liver samples. cMET expression in compensated liver specimens was detected by both immunohistochemistry and Western blot. was significantly reduced compared to isolated control human hepatocytes (P=0.023). 2A and 2B). There was no significant difference in EGFR expression between normal and diseased liver samples. However, there was no significant difference in the expression of EGFR (Figures 2A and 2B). Compared with normal human hepatocytes, phospho-EGFR(Y1086) was significantly increased in patients with decompensated disease. It was highly expressed in hepatocytes derived from diseased livers from individuals with glaucoma (Figures 6A and 6B). Because the continuous cycle of cell death and hepatocyte replication is a hallmark of liver cirrhosis (Tsocha tzis EA et al., 2014), this observation was confirmed in hepatocytes from patients with end-stage liver failure. It has been confirmed that in end-stage hepatocytes, [ phospho-H3(Ser10)] and a reproducible marker of cell death (active caspase 3) Expression was demonstrated.

[0122] In hepatocytes from functionally decompensated livers, cMET inhibits AMPKα and AKT It can be controlled, and cMET is significantly downregulated, indicating that AMPKα The activation process of AMPKα and AKT was analyzed. Total AMPKα, activated AMPKα (T hr172) and its ratio were consistent between hepatocytes with decompensated function and normal human hepatocytes. However, the total AKT, activated AKT, and AKT-dependent AKT activation were not statistically different (Figures 2A and 2B). KT (Thr308), and its ratio, were measured in liver samples from patients with decompensated liver function. and was significantly reduced in isolated hepatocytes (Figures 2A and 2B). The oxidation site (Ser473) was detected in isolated liver samples from normal controls or decompensated specimens. or was unchanged in hepatocytes (Fig. 2A and B).

[0123] To further analyze the relationship between HNF4α, its nuclear localization, and post-translational modifications, Amann's rank correlation test was performed. cMET expression was significantly higher than that of total HNF4α (r = 0.76; P = .001). 0.021; Figure 2C ) and nuclear HNF4α ( r = 0.71; P = 0.037; Figure 2C ). Activated AKT (Thr308) also showed a positive and statistically significant correlation with total HNF4α. (r = 0.73; P = 0.031; Figure 2C ) and nuclear HNF4α (r = 0.82; P = 0.011; Figure 2C), whereas cytoplasmic HNF 4α inhibited cMET (r = −0.80; P = 0.014; Figure 2C) and activated AKT (T hr308) (r = -0.77; P = 0.021; Figure 2C). , the ratio of phospho-AKT (Thr308) / total AKT significantly increased the expression of cMET (r = 0.80; P = 0.014; Figure 6A) and total AKT (r = 0.71; P = 0.037; Figure 6A). Thus, reduced cMET correlates with reduced activation of the AKT pathway, It was associated with reduced HNF4α and higher expression of HNF4α in the cytoplasm.

[0124] [Example 5] Nuclear localization of HNF4α is influenced by the cMET / AKT axis and correlates with the severity of liver dysfunction As can be seen in Figure 3A, pathway analysis revealed a significant correlation between cMET expression and nuclear HNF4α expression. There was a significant causal relationship between the levels of nuclear HNF4α and activation of HNF4α (0.56; P = 0.004). There was a direct relationship between the ratio of AKT(Thr308) / total AKT (0.05; P = 0.0 06). Modeling also revealed that the total HNF4α expression level contributes to its nuclear localization. However, cMET expression was significantly associated with total HN It was negatively associated with F4α expression (−0.37; P = 0.024) (Fig. 3A). MET expression does not directly affect total HNF4α expression, but only its nuclear localization. The level of nuclear expression correlates with the degree of liver dysfunction (Child-Pugh score). To assess whether this is related to the nuclear HNF4α expression level, linear regression analysis was performed. showed that the bell had a significant inverse relationship with the Child-Pugh score (R 2 =0.8 0; P = 0.007) (Figure 3B). Taken together, the pathways for the expression of these proteins and linear regression statistical analysis demonstrated that HNF4α localization was associated with liver disease progression and cM ET expression and AKT phosphorylation maintain the nuclear localization and function of hepatocyte HNF4α. This indicates that the company plays a central role in

[0125] Next, we performed principal component analysis (PCA) to evaluate HNF4α post-translational modifier-related molecules. , molecular patterns correlate with liver function (Child-Pugh score) in end-stage hepatocytes. As shown in Figure 3C, PC1 (69.9 %) and PC2 (30.1%) were associated with liver function in the isolated hepatocytes studied. Identify 100% of the variation in the bell (Child-Pugh score) of normal human hepatocytes The vectors characterized were cMET and the ratio of activated AKT(Thr308) / total AKT. The rate, total HNF4α, and nuclear HNF4α were significantly increased in human hepatocytes, whereas in vivo expression levels of HNF4α were significantly increased in human hepatocytes. The negative features characterized were cytoplasmic HNF4α and active caspase 3 expression ( Figure 3 C and 3D). Taken together, this statistical analysis demonstrates the efficacy of differentiation of human hepatocytes with end-stage liver failure. The molecular profiling confirmed the expression of cMET, activated AKT (Thr308), and total ATP. Establish a causal relationship between mitochondrial and nuclear HNF4α expression.

[0126] [Example 6] Nuclear retention of HNF4α is mediated by decreased acetylation in patients with end-stage liver failure and is reduced. One of the targets of AKT is the activation of CREB-binding protein (Dekker FJ et al., 2009). CREB-binding proteins regulate specific acetylation of nucleosomal histones. activity, which increases the accessibility of transcription factors to nucleosomal DNA and thus the nucleus It is well known that the axis activates transcription and maintenance of transcription factors in the , which may be related to the nuclear retention of HNF4α (Soutoglou E et al., 2000). Global transcriptome and in silico analyses identified the cMET / AKT kinase The ATPase axis pathway regulates HNF4α localization and stability via activation of CREB-binding protein Since it has been shown that acetylated HNF4α can regulate Nuclear expression of HNF4α was measured (Figures 4A-4C), and HNF4α acetylation in the nucleus was significantly elevated compared to normal controls. and found that it was significantly reduced in hepatocytes from patients with end-stage liver failure. Furthermore, the acetylation of HNF4α correlated with the degree of liver dysfunction (challenge). To confirm that the correlation between the mean and mean scores (Hill-Pugh score) was established, a linear regression analysis was performed. The decrease in cetylated HNF4α was directly and significantly correlated with liver dysfunction. (R 2 = 0.71; P = 0.004; Figure 4C). As proof of principle, the nuclear localization of HNF4α To confirm the role of activated AKT (Thr308) in localization, we performed AKT signaling studies. Preliminary experiments were carried out on freshly isolated normal human hepatocytes by inhibiting Isolated normal human hepatocytes were treated with MK-2206, a potent allosteric pan-AKT inhibitor. After 24 hours of AKT inhibitor treatment, activated AKT (Th r308), 80% of nuclear HNF4α, 25% of nuclear HNF4α, and acetylated nuclear HNF4α The expression of 14% was reduced.

[0127] [Example 7] Nuclear retention of HNF4α is regulated by multiple signaling molecules and contributes to end-stage liver disease. It shows a significant negative association with malnutrition. HNF4α is a master regulator of liver function (Babeu JP et al., 2014; Chellappa K et al., 2012;Guo H et al., 2014;Lu H et al., 2016 , Song Y et al., 2015;S outoglou E et al., 2000;Sun K et al., 2007;Xu Z et al., 2007;Zhou W et al., 2 012; Bell AW et al., 2006). Alterations in HNF4α expression may contribute to liver disease with multiple etiologies. associated with, for example, cancer, hepatitis B and C, alcohol-mediated cirrhosis, and NASH. (Babeu JP et al., 2014; Chellappa K et al., 2012; Guo H et al., 201 4;Lu H et al., 2016 , Song Y et al., 2015;Soutoglou E et al., 2000;Sun K et al. al., 2007;Xu Z et al., 2007;Zhou W et al., 2012;Bell AW et al., 2006). T.L. In an animal model with F, a strong reduction in HNF4α expression was identified, and gene therapy By restoring HNF4α production using a treatment, liver cells can be rebooted to normal function. (Nishikawa T et al., 2014). Whether this observation applies to humans remains to be evaluated. To evaluate the efficacy of IFN-γ-glucan in livers of a large cohort of patients with decompensated liver function, We investigated the expression of transcription factors such as HNF4α (Guzman-Lepe J et al., 2018). mRNA levels were downregulated and liver dysfunction was observed based on the Child-Pugh classification. The nuclear localization of HNF4α in these studies was found to be consistent with the degree of There was no gender (Guzman-Lepe J et al., 2018).

[0128] Human hepatocytes were cultured in patients with cirrhosis caused by NASH and end-stage liver disease (Child-Pugh et al., 2014). B, C) Explanted liver tissue from a patient with liver failure and alcohol-mediated Laennec cirrhosis These isolated hepatocytes showed the same activity as normal control hepatocytes. Compared with the control group, cytoplasmic HNF4α was increased and nuclear HNF4α was decreased. In addition, localization of HNF4α to the cytoplasm or nucleus in failing and cirrhotic human hepatocytes was confirmed. These data suggest that the nuclear transport of HNF4α is essential for the development of hepatocyte dysfunction. or that pathways regulating retention may be targets for the treatment of end-stage liver failure. Shows.

[0129] AMPK and AKT kinases are major constituents that can regulate HNF4α localization element (Hong YH et al., 2003;Song Y et al., 2015;Soutoglou E et al., 2000 AMPK is involved in maintaining energy homeostasis and activating adenosine triphosphate (AT P) plays a central role in promoting the production pathway and reducing ATP consumption On the other hand, AKT activation has been shown to affect cell proliferation, survival, and development. Promotes cell proliferation (Manning BD et al., 2017; Morales-Ruiz M et al., 2017). AKT activity Phosphorylation is mediated by phosphorylation at threonine 308 and / or serine 473 ( Praveen P et al., 2016; Inoue J et al., 2017). Activated AKs in end-stage human hepatocytes The T(Thr308) level was significantly decreased, indicating that activated AKT(Thr308) and These findings demonstrate a significant correlation between HNF4α localization and We have shown that AKT phosphorylation at Thr308 contributes to hepatocyte failure in the end-stage of liver disease. This indicates that the technology may play an important role in

[0130] Analysis was performed on cMET and EGFR. These two central receptors are involved in A Upstream regulator of KT and AMPK, involved in liver function and regeneration (Nataraja n A et al., 2007;Komposch K et al., 2015;Paranjpe S et al., 2016;Tsagianni A et al., 2018; Alam A et al., 2017). Disruption of cMET and EGFR in mice The combination of these alters hepatic homeostasis and leads to end-stage liver failure (Tsagianni A et al., 2018). Reduced cMET expression in failing cirrhotic human hepatocytes, and There was decreased expression that directly correlated with HNF4α localization. In contrast, in human cirrhotic tissue, There was no difference in total EGFR expression in either treated or control human hepatocytes. Thus, in human liver, cMET mediates AKT pathway activation and HNF4α localization. Furthermore, different statistical analyses ( Spearman's rank correlation test, pathway analysis, linear regression analysis, and principal component analysis) The relationship between post-translational modifiers analyzed in human failing, cirrhotic and normal hepatocytes was investigated. These statistical analyses allow the establishment of correlations. 8) revealed that the expression level of HNF4α in the nucleus is directly related to the expression level of cMET protein. The negative association found between cMET expression and total HNF4α suggests that cMET expression is associated with increased expression of total HNF4α. This indicates that it does not directly affect the expression of the protein, but only its nuclear localization.

[0131] Acetylation of HNF4α promotes activation of AKT (Th) in failing and cirrhotic human hepatocytes r308) and increased transcription factor binding to nucleosomal DNA. Direct binding of AKT to CREB-binding protein, a molecule with intrinsic acetylation activity Based on this effect, it can be affected in cirrhotic and failing liver cells. Strikingly, F4α acetylation was reduced in human hepatocytes from cirrhotic livers, and its These observations were supported by the results of the analysis of the threonine 308 level. AKT phosphorylation regulates CREB-binding protein through acetylation It has been shown that this mediates the nuclear retention of HNF4α (Soutoglou E et al., 2000).

[0132] In summary, the localization of HNF4α in the cytoplasm is consistent with the nuclear localization of HNF4α during the progression of liver disease. This results from alterations in the molecular pathway that maintains F4α, cMET, and activated AKT (Thr30 8) was down-regulated and affected the acetylation and nuclear retention of HNF4α. These data suggest that nuclear localization of HNF4α may contribute to hepatocyte function in chronic liver disease. indicates recovery.

[0133] [Example 8] Transduction of primary human hepatocytes with lentiviral (LV) constructs of transcription factors. Transduction of primary human hepatocytes with the transcription factor LV. Hepatocytes were transduced with LV to prevent hepatocyte dedifferentiation. The cells are then cultured in a double collagen (thick layer) system. The protocol will be used. Prepare the following: WARM: dPBS, HMM (basal + Si SingleQuots), HCM (HBM basal + HCM SingleQuots); on ice : Green fluorescent protein (GFP) LV * , transcription factor (TF) LV *, Max Enhan cer, TransDux; Others: 1.5mL tubes, 50mL tubes, tips P, pipette.

[0134] 1. Plate 5e5 hepatocytes per well onto a thick layer of collagen and Leave it on for 4 hours.

[0135] 2. Wash the wells twice with warm dPBS to remove dead cells and replace the medium with 500 μL of HMM. (without FBS) for replacement.

[0136] 3. Prepare five tubes and label them as follows: a. GFPLV-2; b. GF PLV-10;c.TFLV-0;d.TFLV-2;e.TFLV-10.

[0137] 4. Add 50 ml of HMM / Max Enhancer / TransDux (HMT) solution. L tube: 12.323 mL HMM + 3.100 mL Max Enhancer Prepare with +77.5uL of TransDux.

[0138] 5. Dispense the LV solution into pre-labeled tubes: a. GFPLV-2: 618.3 uL of H a. MM + 1.7uL GFP LV; b. GFPLV-10: 611.6uL HMM + 8 a. 4uL of GFP LV; c. TFLV-0: 620uL of HMM only; d. TFLV- 2:618.6uL HMM + 1.44uL TF4LV; e.TFLV-10:612 Prepare with 0.82uL HMM + 7.18uL TF4LV.

[0139] 6. Replace the HMM medium in the wells with 500 μL of HMT solution.

[0140] 7. Dispense 100 uL of each LV solution into the wells. Swirl the plate to mix.

[0141] 8. The next day, wash the wells twice with warm dPBS to remove dead cells and residual LV solution. do.

[0142] 9. Layer a thick layer of collagen on the cells and allow the collagen to gel for 2 hours.

[0143] 10. Add 500 uL of HCM to the wells and replace with fresh HCM daily.

[0144] 11. Obtain samples at 72 and 96 hours post-transduction (refer to the protocol at 72 hours) reference).

[0145] To obtain the sample at 12.96 hours, wash the cells with warmed dPBS and add 500 μl of medium. Replace with L HCM (without FBS).

[0146] Notes: * Thaw the LV quickly in a water bath at 37°C. Transfer to the hood and thaw by rotating, inverting, or Mix by gentle vortexing or stirring and keep on ice. Coat the cells and refrozen them at -80°C. Each refrozen batch will lose 10-20% of the virus activity. do.

[0147] Transcription factor (TF) LV constructs. The TF LV constructs were: PROX1, NR5A2, NR0B2, MTF1, and SREBP1 a polynucleotide encoding EP300, POM121C, or HNF4α; indicates RNAi corresponding to DNAJB1 / HSP40, ATF6, ATF4, or PERK. A lentiviral vector (Systems Bio) containing a polynucleotide encoding (Oscience, catalog number CS970S-1).

[0148] Conditioned media is collected for ELISA (72 and 96 hours). Two cultures per group are used. Collect 1200 μL of conditioned medium from the well and transfer to a 1.5 mL tube. Replace with warmed HMM. Centrifuge the conditioned medium at 20,000 x g for 2 minutes. Transfer to a new tube and store at -20°C.

[0149] Take photos of GFP expression and bright field to determine transfection efficiency (72 and (and 96 hours). Wash the floating cells with warmed dPBS. Replace with warmed HCM. Filter 2 (green excitation) for GFP expression and filter 6 for bright field. Take a photo.

[0150] Collect cell lysates in Qiazol for RNA extraction (72 and 96 h) Wash cells twice with warm dPBS. Coat wells with 600 μL of Qiazol. Incubate for 1 minute. Use a P1000 to scrape the cells off the plate. Transfer to a 5 mL tube. Store at -20 °C until RNA isolation.

[0151] Fix wells for IF (72 hours). Wash cells twice with warm dPBS. Coat the wells with 0 μL of 4% PFA solution and incubate for 40 minutes. Wash the plate three times with 1 mL of dPBS, 10 min per wash. Add 1 mL of dPBS. Store at 4 °C until staining for HNF4A.

[0152] Collect cell lysates for Western blot (72 and 96 hours). Prepare ice-cold lysis solution containing:

[0153] [Table 3]

[0154] Wash the cells twice with warm dPBS. Coat the wells with 200 μL of ice-cold lysis solution. Incubate in a walk-in refrigerator with rocking for 30 minutes. Using a lysing machine, cells were detached and the lysate containing the precipitate and solids was collected and pre-labeled. Transfer to a belled tube. Spin at 20,000 x g for 10 minutes at 4°C. Transfer to a pre-labeled 1.5 mL tube. Store the lysate in a -80°C freezer.

[0155] TF immunofluorescence co-staining (in a 12-well plate with a thick collagen sandwich layer) (If necessary, gently aspirate the sample to prevent cell detachment. Allow the sample to dry. do not have. * Spin at maximum speed for 5 minutes. * The secondary antibody may be replaced with another suitable antibody depending on the experiment. It can be replaced.

[0156] Fixation (if cells are already fixed, proceed to blocking and permeabilization steps) The samples were fixed with 4% paraformaldehyde in PBS, pH 7.4, for 40 minutes at room temperature. Wash the samples three times with ice-cold PBS for 10 minutes each wash. Proceed to staining or Store at 4°C until staining (maximum 2 weeks).

[0157] Blocking and permeabilization. Samples are washed twice with 1 mL of PBS. Wash buffer (PBS, 0.1% BSA, and 0.1% Tween) three times, with The samples were then washed for 10 minutes with 1 mL of blocking buffer (PBS, 10% normal donkey blood). supernatant, 1% BSA, 0.1% Tween, and 0.1% Triton X-100) Block and permeabilize by incubating at 4°C for 2 hours.

[0158] Antibody incubation: Add mouse anti-TF stock 1° Ab by vortex mixing and Centrifuge * A 1:500 dilution of mouse anti-TF1 Ab was prepared in blocking buffer. Coat the sample with 600 μL of diluted 1°Ab and incubate in a humidified chamber. Incubate for 6 hours at room temperature or overnight at 4°C. 1. Aspirate the Ab solution and wash the cells. Wash three times with buffer, 10 min each wash. Vortex and centrifuge the solution (i.e., ribosomal nucleotide A21203). * 1:250 rare Prepare diluted 2°Ab in blocking buffer. Test with 600 μL of diluted 2°Ab. Coat the plates with the material and incubate them in a humidified chamber at room temperature for 2 hours. The Ab solution is aspirated and the cells are washed three times with wash buffer, 10 minutes per wash.

[0159] Counterstaining and mounting: Wash the samples three times with PBS. Incubate for 2 minutes in 1 mL of 1 μg / mL Hoechst 33342. Wash three times with 0.5% CO. Samples may be stored in the dark at 4°C. Use the RED channel to Test F.

[0160] [Example 9] Transfection of primary human hepatocytes with transcription factor (TF) mRNA (50, 100, 500 ng) infection Transfection of primary human hepatocytes. Preheat the following: DPBS, HMM ( HMM basal medium + HMM SingleQuots), Opti-MEM → RT; on ice: Lipofectamine Messenger Max → RT, mRNA → RT; Others: 1.5 mL Prepare tubes, 50 mL tubes, tips, and pipettes.

[0161] 1. Wash the cells with warm DPBS and replace the medium with 500 μL of HMM (without FBS). .

[0162] 2. Prepare two sets of tubes labeled as follows: a. GFP-50. b. GFP-100. c.GFP-500. d.TF-0. e.TF-50. f.TF-10 0. g.TF-500. TF is PROX1, NR5A2, NR0B2, MTF1, SR Selected from EBP1, EP300, POM121C and HNF4α.

[0163] 3. Prepare a second diluted mRNA in Opti-MEM: a. DilGFP: 3 2.5 uL OptiMEM + 3.61 uL GFP mRNA. b.DilHNF:4 0.3uL OptiMEM + 4.47uL TF mRNA.

[0164] 4. In the first set of pre-labeled tubes, add the mRNA-OptiMEM mix. Prepare: a. GFP-50: 312.2 uL Opti-MEM + 2.8 uL Di lGFP. b. GFP-100:309.4uL Opti-MEM+5.6uL Di lGFP. c.GFP-500:287.3uL Opti-MEM+27.7uL D ilGFP. d.HNF-0:315uL Opti-MEM. e.TF-50:311 0.5uL Opti-MEM + 3.5uL DiTF. f.TF-100:308.1 uL Opti-MEM + 6.9 uL DilTF. g.TF-500: 280.7 uL of Opti-MEM + 34.3uL of DilTF.

[0165] 5. Prepare diluted Lipo mix: a. 2, 220.75 uL of Opti-M EM+141.75uL Lipo.

[0166] 6. Vortex the diluted Lipo mix for 2-3 seconds and add it to the second set of pre-labeled Dispense 310uL into the tube. Incubate at room temperature for 10 minutes.

[0167] 7. Add 310uL of each mRNA-OptiMEM mix to the diluted Lipo mix Transfer to the tube containing 100 μl of PBS. Incubate at room temperature for 5 minutes.

[0168] 8. Dispense 100uL of each mix into each well.

[0169] 9. Incubate and the next morning, wash with warm dPBS and add 500 μL of HMM (without FBS) ) to replace it.

[0170] 10. Transfection of 24 * and 48 * After 2 hours (hepatocyte plating The sample is obtained at the time (depending on when the sample is to be obtained).

[0171] Conditioned media is collected for ELISA (24 and 48 hours). Two cultures per group are used. Collect 1200 μL of conditioned medium from the well and transfer to a 1.5 mL tube. Replace the medium with warm HMM. Centrifuge the conditioned medium at 20,000 x g for 2 minutes. Transfer the supernatant to a fresh Transfer to a clean tube and store at -20°C.

[0172] Take photos of GFP expression and bright field to determine transfection efficiency (24 and The floating cells were washed with warm dPBS and replaced with warm HMM. Photographs were taken using filter 2 (green excitation) and filter 6 for bright field. do.

[0173] Collect cell lysates in Qiazol for RNA extraction (24 and 48 h) Wash cells twice with warm dPBS. Coat wells with 600 μL of Qiazol. Incubate for 1 minute. Use a P1000 to scrape the cells off the plate. Transfer to a 5 mL tube. Store at -20 °C until RNA isolation.

[0174] Fix wells for IF (24 hours). Wash cells twice with warmed dPBS. Coat the wells with 750 μL of 4% PFA solution and incubate for 20 minutes. Wash the wells three times with 1 mL of dPBS, 5 minutes per wash. Add 1 mL of dPBS. Store at 4°C until stained for TF.

[0175] Collect cell lysates for Western blot (24 and 48 hours). Prepare ice-cold lysis solution containing:

[0176] [Table 4]

[0177] Wash the cells twice with warm dPBS. Coat the wells with 200 μL of ice-cold lysis solution. Incubate in a walk-in refrigerator with rocking for 30 minutes. Using a lysing machine, cells were detached and the lysate containing the precipitate and solids was collected and pre-labeled. Transfer to a sealed tube. Spin at 20,000 x g for 10 minutes at 4°C. Clear the supernatant. Transfer to a pre-labeled 1.5 mL tube. Store the lysate in a -80°C freezer. .

[0178] Immunofluorescence co-staining (staining, fixation, blocking and permeabilization, antibody incubation) , and counterstaining and mounting) were as described above in Example 9.

[0179] [Example 10] Transcription factors and regulators PROX1, NR5A2, NR0B2, MTF1, SREBP1 , EP300 and POM121C promote H in cirrhotic hepatocytes with end-stage liver failure Improves nuclear expression of NF4α. Liver-enriched transcription factors are stably expressed in hepatocytes from rats with end-stage liver cirrhosis. One of these is hepatocyte nuclear factor 4 alpha (HNF 4α) both in culture and in vivo. It was found that damaged liver cells were reprogrammed and their function was restored. Progressive liver disease The level of HNF4α mRNA expression in diseased liver in a large cohort of patients with was correlated with the degree of liver dysfunction (Child-Pugh classification), and As in previous studies, the expression was not localized in the nucleus. In the livers of patients with cirrhosis, the RNA expression level of HNF4α decreased with the decline in liver function. These findings suggest that the expression of β-glucan in the liver is reduced by β-glucan and that the protein expression is found in the cytoplasm. This could explain the impaired liver function in patients with HIV. HNF4α and other transcription factors / regulators-associated pathways involved in nuclear protein translocation is downregulated in cirrhotic hepatocytes from patients with end-stage liver failure, in which case In this study, the nuclear level of HNF4α was significantly reduced and the cytoplasmic expression of HNF4α was increased. In addition, four major transcription factors responsible for endoplasmic reticulum (ER) stress were identified. The study found that regulatory factors were significantly upregulated. Manipulation of HNF4α and pathways involved in hepatocellular function in patients with end-stage liver failure This shows that the

[0180] PROX1, NR5A2, NR0B2, MTF1, SREBP1, EP300 and P OM121C protein expression correlates with the degree of liver dysfunction in patients with end-stage liver failure. HNF4α must be expressed in the nucleus to function properly; therefore, In addition, we investigated the signaling pathway involved in the nuclear localization of HNF4α by examining the function of exogenous HNF4α. RNA sequencing analysis identified transcription factors and regulatory Factors PROX1, NR5A2, NR0B2, MTF1, SREBP1, EP300 and The identification of POM121C as a key modulator of HNF4α Antibody-based assays for these molecules are being used to treat NASH or alcohol-induced cirrhosis. Primary human hepatocytes (Child-Pugh "B" and "C" hepatocytes) isolated from livers of patients undergoing liver transplantation were used. and "C") or normal control hepatocytes. HNF4α expression was measured by Western blot analysis. As measured by ELISA (Figure 8A), decompensated liver cells exhibited significantly higher levels of IL-12 than isolated control human hepatocytes. There was also a significant difference in MTF1 expression as liver failure progressed. Furthermore, using simple linear regression, the Child-Pugh-scored human Hepatocytes were correlated with the protein expression of HNF4α and MTF1, suggesting that HNF Both 4α and MTF1 were found to be significantly correlated with the degree of liver failure (p=0 .007) (Figures 8B-8F). In addition, the protein expression of NR0B2 (Figures 9A-D), N Protein expression of R5A2 (Figure 10A-D), protein expression of PROX1 (Figure 11A-D) D) but their expression was significantly lower in hepatocytes than in Child-Pugh C. It was found that the degree of cell dysfunction correlated with the

[0181] These identified transcription factors and regulators of HNF4α nuclear expression and localization To further understand the role of α-amyloid in the expression of α-amyloid in human hepatocyte cell lines, we used CRISPR / Cas9 to By gene editing, PROX1 or NR5A2 or NR0B2 or MTF1 or Knockout of either SREBP1 or EP300 and POM121C expression ( KO) (Figure 15A-B). PROX1, NR5A2, NR0B2, MTF1, SRE KO of BP1, EP300, or POM121C significantly reduced nuclear expression of HNF4α. It was found that a significant reduction occurred when PROX1 or SREBP1 was knocked out (FIG. 15A). Particularly high non-nuclear expression of HNF4α was observed in human livers with end-stage liver failure. Previous studies in cells have also identified cytoplasmic expression of HNF4α. Furthermore, the effect of HNF4α alone or PRO on the induced nuclear expression of HNF4α was investigated. X1 or NR5A2 or NR0B2 or MTF1 or SREBP1 To test the effect of HNF4α in combination with either POM121C or HNF4α, liver transplantation was performed. Human liver tissue isolated from explanted livers of patients with end-stage liver failure due to NASH who underwent transplantation Treatment of hepatocytes was performed (Figure 16). After 6 hours, an approximately 1-fold increase in nuclear expression of HNF4α was found compared to the control. However, HNF4α-AAV processing was not affected by PROX1-AAV or is NR5A2-AAV or NR0B2-AAV or MTF1-AAV or SREB When combined with either P1-AAV or POM121C-AAV, all combinations The combination, especially if the combination includes PROX1 or SREBP1 in addition to HNF4α, It significantly induced the nuclear expression of NF4α (Fig. 16).

[0182] Therefore, this study investigated the transcription factors and regulators PROX1, NR5A2, and NR0B2 , MTF1, SREBP1, EP300 and POM121C with end-stage liver failure We demonstrate that this improves the nuclear expression of HNF4α in cirrhotic liver cells. The results suggest that HNF4α is linked to one or more transcription factors and regulators, such as PROX1 or or NR5A2 or NR0B2 or MTF1 or SREBP1 or EP300 and All combinations containing HNF4α along with POM121C and POM121C significantly increased nuclear expression of HNF4α and its regeneration. Enhanced programming capabilities are demonstrated to treat end-stage liver failure.

[0183] References: [Table 5-1] [Table 5-2]

Table 5-3

Table 5-4

Table 5-5

Table 5-6

Table 5-7

Claims

1. A method of treating liver disease in a subject in need thereof, comprising administering to said subject a composition and administering to the patient a composition comprising: PROX1, NR5A2, NR0B2, MTF1, S One or more selected from the group consisting of REBP1, EP300, and POM121C A method for increasing the amount or function of a number of transcription factors.

2. The composition is a vector, and the vector is a vector encoding PROX1, NR5A2, NR0B2 , MTF1, SREBP1, EP300, and POM121C. The method of claim 1, comprising one or more nucleic acids encoding:

3. The composition is a vector, and the vector is a vector encoding PROX1 and / or SREBP.

10. The method of claim 1, comprising one or more nucleic acids encoding .

4. Any of claims 1 to 3, wherein the one or more nucleic acids are DNA or mRNA.

10. The method according to claim 1.

5. said administering said composition increases the amount of HNF4α in the nuclei of hepatocytes in said subject. The method according to any one of claims 1 to 4.

6. 10. The method of claim 1, wherein the administration of the composition does not increase the total amount of HNF4α in the hepatocytes.

6. The method according to any one of claims 5 to 5.

7. 6. The method of claim 1, wherein the administration of the composition increases the total amount of HNF4α in the hepatocytes.

10. The method according to any one of the preceding claims.

8. Any of claims 1 to 7, wherein the vector further comprises a nucleic acid encoding HNF4α.

10. The method according to claim 1.

9. further comprising administering to the subject a vector comprising a nucleic acid encoding HNF4α. The method according to any one of claims 1 to 7.

10. 10. The nucleic acid of claim 8 or 9, wherein the nucleic acid encodes HNF4α isoform 2 (P1). The method described.

11. The method of claim 8 or 9, wherein the nucleic acid encoding HNF4α comprises SEQ ID NO:

1. Law.

12. 12. The method according to claim 1, wherein the liver disease is liver fibrosis, liver cirrhosis, liver cancer, or end-stage liver disease.

10. The method according to any one of the preceding claims.

13. The method according to any one of claims 1 to 12, wherein the liver disease is cirrhosis.

14. The method of any one of claims 1 to 13, wherein the subject is a human.

15. A method of treating liver disease in a subject in need thereof, comprising administering to said subject a composition and administering to the subject a composition comprising DNAJB1 / HSP40, ATF6, ATF4, and and PERK, or Or a method for suppressing its function.

16. The method of claim 15 , wherein the composition is a nucleic acid.

17. 17. The method of claim 16, wherein the nucleic acid is DNA or RNA.

18. said administering said nucleic acid increases the amount of HNF4α in the nuclei of hepatocytes in said subject; The method according to any one of claims 15 to 17.

19. 15. The method of claim 14, wherein said administering said composition does not increase the total amount of HNF4α in said hepatocytes.

19. The method according to any one of claims 1 to 18.

20. 15 to 17, wherein the administration of the composition increases the total amount of HNF4α in the hepatocytes.

20. The method of any one of 19.

21. 21. The composition according to claim 15, wherein the composition further comprises a nucleic acid encoding HNF4α.

1. The method according to claim 1.

22. further comprising administering to the subject a vector comprising a nucleic acid encoding HNF4α. The method according to any one of claims 15 to 21.

23. 23. The nucleic acid of claim 21 or 22, wherein the nucleic acid encodes HNF4α isoform 2. How to do it.

24. 23. The method of claim 21 or 22, wherein the nucleic acid encoding HNF4α comprises SEQ ID NO:

1. How to do it.

25. 15 to 2, wherein the liver disease includes liver fibrosis, liver cirrhosis, liver cancer, or end-stage liver disease.

5. The method according to any one of claims 4 to 4.

26. The method of any one of claims 15 to 25, wherein the subject is a human.

27. A composition comprising a vector, wherein the vector is selected from the group consisting of PROX1, NR5A2, NR0B 2, MTF1, SREBP1, EP300, and POM121C, and their functional one or more fragments encoding one or more transcription factors selected from the group consisting of A composition comprising a nucleic acid.

28. The combination of claim 27, further comprising another vector comprising a nucleic acid encoding HNF4α. Finished product.

29. 29. The composition of claim 28, wherein the nucleic acid encoding HNF4α comprises SEQ ID NO:

1.

30. 1. A method of treating liver disease in a subject in need thereof, comprising administering to said subject HNF4α A method comprising administering a vector comprising a nucleic acid encoding isoform 2.

31. 31. The method of claim 30, wherein the nucleic acid comprises SEQ ID NO:

1.

32. 30. The method of claim 30, wherein the liver disease is liver fibrosis, liver cirrhosis, liver cancer, or end-stage liver disease.

31. The method according to claim 31.

33. 33. The method of claim 32, wherein the liver disease is cirrhosis.

34. The method of any one of claims 30 to 33, wherein the subject is a human.

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